Chapter 4

Results and Discussion
RESULTS AND DISCUSSION

In developing as well as underdeveloped countries, the industrial
effluents are released directly or indirectly into natural water resources, mostly
without proper treatment, thus posing a serious threat to the environment
(Altug and Balkis, 2009). Environmental pollution is an extremely important
issue today, affecting all of us in one way or the other. Due to rapid increase in
human population and industrialization, the demand for natural raw materials
and source of energy are increasing day by day (Abhay and Rajput, 2009).
Many rivers of the world receive flux of sewage, domestic waste, industrial
effluents and agricultural waste which contain substances varying from simple
nutrients to highly toxic chemicals (Benazir et al., 2010). Tannery industry
contributes significantly towards exports, employment generation and occupies
an important role in Indian economy. Heavy metals released from tanneries are
kept under environment pollutant category due to their toxic effects on plants,
animals and human beings. They interfere with physiological activities of plants
such as photosynthesis, gaseous exchange and nutrient absorption and cause
reduction in plant growth, dry matter accumulation and yield (Sharma and
Agrawal, 2005). They cause direct toxicity, both to human and other living
beings due to their presence beyond specified limits. Heavy metal pollution of
soil and waste water is a significant environmental problem and has a negative
impact on human health and agriculture (Michalak, 2006).
The reuse of waste waters and industrial effluents for irrigation to
crop plants after proper dilution is an useful technique (Rehman et al., 2007).
Tannery effluent can be diluted and reused for agriculture purpose which can
also act as a good fertilizer (Mariappan and Rajan, 2002).
In the selected area of our study the continuous discharge of tannery
waste water has polluted the water and lands of the nearby villages namely
Chinnalapatti, Begampur, Kottapatti and many other places. The potable water
Effect of tannery effluent on water and soil profile, plant growth and human health
51
of the residential area was found to be salty and polluted and soil properties of
the cultivable land of the nearby area was also affected. The negligence of the
safe disposal of tannery wastes had led to deleterious effects on the biosphere
as a whole. Hence, in the present study, the impact of the tannery effluent on
water, soil, plant and human beings were assessed. The results of the study
entitled Effect of tannery effluent on water and soil profile, plant growth
and human health are presented and discussed as follows:
PHASE I
4.1 Characterization of the tannery effluent, target area water and soil
samples
4.1.1 Characterization of tannery industry effluent
4.1.2 Biochemical profile of the target area water samples
4.1.3 Physicochemical characteristics of target area soil samples
PHASE II
4.2 Growth studies of selected plants using diluted tannery effluent
4.2.1 Biometric observations
4.2.2. Biochemical parameters
4.2.3 .Histochemical observations of root samples of the selected plants
4.2.4. Yield parameters
PHASE III
4.3 Health profile of the tannery industry workers
4.3.1 Hematological parameters
4.3.2 Assessment of the liver function
4.3.3 Assessment of the renal function
4.3.4 Assessment of metal contents
52
PHASE I
4.1 Characterization of tannery effluent, target area water and soil samples
4.1.1 Characterization of tannery industry effluent
Tannery industry contains several organic and inorganic chemicals,
which are toxic metals and they cause soil and ground water pollution. These
chemicals cause adverse effect on plant growth and the health of animals and
people living in that area. Processing of hides and skin to leather uses many
chemicals at various stages and hence releases many toxic substances at each
stage. The overall tanning process performed in drums can be characterized by
a high consumption of water and chemicals with collagen. Chemicals are added
in excess and are only partly taken up by the leather and the remaining is
released in the effluent (Scholz and Lucas, 2003).
Heavy metals can pose health hazards if their concentrations exceed
allowable limits. Even when the concentration of metals does not exceed these
limits there is still a potential for long-term contamination, and heavy metals are
known to accumulate within biological system (Altaf et al., 2008). Hence the
effluent released is expected to have a higher amount of chemicals and toxic
metals.
In every step of tanning process a considerable amount of waste water is
released. The waste water was found to contain salts, fat, protein and
preservatives for soaking, lime, ammonia and sulphides for fleshing, trimming
and bating, chromium salts and polyphenolic compounds for tanning and
dye and solvent chemicals with metals for wet finishing. Hence tanneries
that perform the complete tanning process produces a complete tanning
mixed waste water. In this view, in the present study, the combined tannery
effluent
was
collected
and
characterized
for
certain
physicochemical
parameters. The results of the physical parameters of the raw effluent

are presented in Table 6.
53
TABLE 6
PHYSICAL PARAMETERS ASSESSED IN RAW TANNERY EFFLUENT
RAW
EFFLUENT
BIS LIMITS
IS 2490-2009
Colour
Brown
Odour
Offensive
Turbid
pH
10.5
5.5 - 9.0
Total hardness (mg/l)
5400
100
24,500
NM
650
100
17,150
2100
PARAMETERS
Turbidity
Electrical conductivity (mg/l)

Total suspended solids (mg/l)
Total dissolved solids (mg/l)
Values are mean of triplicates
NM - Not mentioned
BIS - Tolerance limits for industrial effluent discharged into inland surface waters prescribed by
the Bureau of Indian Standards (2009)
The effluent released from tannery industry was brown in colour and had
an offensive odour. The colour of the effluent might be due to the presence of
biodegradable
and
nonobiodegradable
high
molecular
weight
organic
compounds and high amount of chemicals used during the processing and the
odour may be due to the processing of skin and hides by soaking and liming.
The yellowish brown colour might be hindering the penetration of sunlight
causing depletion in the rate of oxidation process (Ravibabu et al., 2007) and
this colour might be due to physico chemical treatments (Zahid et al ., 2006).
Turbidity is an expression of the optical property that causes light to
be scattered and absorbed rather than transmitted with no change in direction
or flux level through the sample (APHA, 2005). The turbidity of the effluent
might be due to the discharge of high concentrations of carbonate,
bicarbonate, chloride, calcium, magnesium and sodium used in tanning industry
(Chakrapani, 2005).
54
pH was recorded as 10.5, which was above the tolerance limit of

5.5 - 9.0 prescribed by the Bureau of Indian Standards (2009). The higher pH of
the effluent indicates the basic nature of the effluent. The pH of waste water
could vary due to the presence of various tanning and colouring materials.
According to Fadali et al.(2005), the pH of the tannery effluent assessed by
them was found to be 10.0, which was above the permissible limits and they
suggested that this alkaline pH of the effluent could affect the biological property
of the receiving water body.
Total hardness of the tannery effluent in the present study was found to
be 5400 mg/l. Hardness is an indication of calcium and magnesium found in
higher concentration in effluent.
The higher electrical conductivity value of the effluent indicates that
the discharge of chemicals as cations and anions were higher in the waste
water. The higher conductivity alters the chelating properties of water
bodies and creates an imbalance of free metal availability for flora and
fauna (Akan et al., 2008). Venkatesh et al. (2009) recorded that the electrical
conductivity, pH, chloride, sulfides, biological oxygen demand and chemical
oxygen demand in tannery effluent were much higher than the tolerance limits
for industrial effluent discharged into land surface.
Levels of total suspended solids (TSS) found in the effluent (650mg/l)
were greater than that of the permissible limit (100 mg/l).
Somnath (2003)
reported that larger solid particulate matter remains suspended as a result of

charges on the surface of small particles in the effluent.
The effluent showed a higher level of total dissolved solids (TDS)
(17,150 mg/l) . The value was much greater than the tolerance limits (2100 mg/l)
prescribed by Bureau of Indian Standards. Total dissolved solids are mainly
due to carbonates, bicarbonates, chlorides, sulphates, phosphates, nitrates,
nitrogen, calcium, sodium, potassium and iron (Kannan et al., 2009). In the
55
liming section of tanning process, protein, hair, skin and emulsified fats are
removed from the hides, which are released in the effluent and therefore
increase the total solids (Bhalli and Khan, 2006).
Table 7 indicates the chemical characteristics studied in tannery effluent.
TABLE 7
CHEMICAL CHARACTERISTICS OF RAW TANNERY EFFLUENT
RAW
EFFLUENT
BIS LIMITS
IS 2490-2009
Chemical Oxygen Demand
3180
250
Biological Oxygen Demand
1300
30
Carbonate
9850
600
Bicarbonate
10423
NM
Calcium
1440
200
Magnesium
432
30-100
Chloride
5100
1000
Sodium
2300
NM
600
NM
Fluoride
1.0
2.0
Nitrate
440
100
Nitrite
32
10
Sulphate
1080
1000
Chromium
19.3
2.0
Nickel
5.5
3.0
Zinc
10.8
1.0
Cadmium
4.2
2.0
PARAMETERS
Potassium
mg/l

NM - Not mentioned
BIS - Tolerance limits for industrial effluent discharged into inland surface waters prescribed
by the Bureau of Indian Standards (2009)
56
Chemical oxygen demand (COD) is the test used to measure pollution

of domestic and industrial waste (Chavan and Wagh, 2005). It is the amount of
oxygen required for the oxidation of inorganic matter using a strong chemical
oxidant. COD is tested to determine the degree of pollution in the effluent
samples (Bhalli and Khan, 2006). COD values in the effluent were found to be
12 times higher than that of the tolerance limits.
Biological oxygen demand (BOD) is the parameter which is widely
used to determine the pollution load of waste water (Chavan and Wagh, 2005).
It is the amount of organic matter in the water and the amount of oxygen
required by the micro organisms to stabilize the biologically decomposable
organic matter in wastes under aerobic conditions (Bhalli and Khan, 2006).
BOD of tannery effluent was found to be 1300 mg / l which was higher than that
of the BIS limits (30 mg/l).
Akan et al. (2007) also reported higher levels of BOD, COD and TSS in
tannery effluent. Industrial effluents and municipal sewage carry organic and
inorganic substances which utilize dissolved oxygen resulting in oxygen
depletion (Ravindran and vasudevan, 2008). Nearly 70% of the emission loads
of biochemical oxygen demand (BOD), chemical oxygen demand (COD)
and total dissolved solids (TDS) emanate from the pretanning operations
(Calherios et al., 2008b).
High carbonate and bicarbonate contribute to the total alkalinity of the
sample (Balakrishnan and Karruppusamy, 2005). Carbonate and bicarbonate of
the effluent were found to be 9850 mg/l and 10423 mg/l respectively. Usage of
sodium bicarbonate during the process of pickling in tannery industry might
have caused the excessive amounts of these in the effluent.
The cations calcium and magnesium present in the effluent were found
to be at higher levels (1440 mg/l and 432 mg/l respectively) compared to BIS
limits. Calcium and magnesium contribute to the hardness of the water and it
57
imparts unpleasant odour, when present in higher levels (More et al., 2002). The
tannery effluent contains fairly large amount of calcium and magnesium
because lime is used for loosening the hair.
Chloride is an indicator of pollution when present in higher
concentrations (Singh et al., 2009). Sodium chloride used as a dehydrating and
antiseptic agent is the source of chloride (Mehdi, 2005). The level of chloride in
the effluent (5100 mg/l) was 5 fold higher than that prescribed by BIS (2009).
The presence of very high amounts of chloride and sulphate is responsible
for high hardness and further it increases the degree of eutrophication
(Kannan et al., 2005).
The level of sodium and potassium in the effluent were 2300 mg/l and
600 mg/l respectively. Sodium sulphide is used in the liming process of hide and
skin. The residual sulphide in the range of 100 200 mg/l goes in the discharge
and causes serious environmental problem (Ram and Roger, 2004). The high
concentrations of sodium and chloride in the effluent were mainly due to the use
of huge salts in different stages of tanning process (Zahid et al., 2006).
Fluoride levels in the effluent were lesser (1.0 mg/l) than that of Bureau
of Indian Standards (2.0 mg/l). Very high nitrate content (440 mg/l) was present
in tannery effluent than the standard value. Nitrite content in tannery effluent
(32 mg/l) was also above the permissible limits (10 mg/l). Waste generated from
tanning generally contains much higher concentration of total dissolved solids
(TDS), total suspended solids (TSS), phenols, chromium, chlorides, nitrates,
nitrites, ammonia and heavy metals. Nitrate is the highest oxidized form of
nitrogen and causes blue baby disease when consumed in excess (Das et al.,
2010)
Sulphate is one of the important anions present in natural water and
produces laxative effect when exceeds the limit (Kasthuri et al., 2005). Sulphate
levels in the effluent were found to be 1080 mg/l. The tolerance limits prescribed
by BIS (2009) for sulphate was 1000 mg/l which was lesser than the amount
58
present in effluent. In a similar study done by Zahid et al. (2006), extremely

high concentrations of sodium, magnesium, chloride, calcium, sulphate and
bicarbonate were reported in tannery effluent. It was also reported by Sheela
and Peethambaram (2007) that excess amount of sulphate, nitrogen and
dissolved solids were present in the tannery effluent.
Chromium level in raw effluent was found to be 19.3 mg/l which was 10
times higher than the amount prescribed by BIS (2.0 mg/l). Chromium is the
major chemical used in tanning process and hence its discharge in the effluent
was found to be high. Continuous discharge of chromium in low concentration
has been reported to be toxic to aquatic life and has been shown to disrupt the
aquatic food chain (Fent, 2004). Extremely high concentrations of chromium,
sodium, magnesium, calcium and ammonia were detected in the tannery
effluent in a study conducted by Krantz and Kifferstein (2002).
Nickel, zinc and cadmium in the effluent were 5.5 mg/l, 10.8 mg/l and
4.2 mg/l respectively. All these metals were present in higher concentrations
compared to the prescribed limits of BIS (2009) (3.0 mg/l, 1.0 mg/l and 2.0 mg/l
respectively). The tannery wastewater is being contaminated with higher levels
of metals (iron, nickel, chromium, zinc, cadmium, manganese and copper) and
their irrigation contaminates the soil vegetables and crops, which when
consumed causes serious health hazards to the consumer (Mohanta et al.,
2010). The presence of cadmium and other heavy metals in the environment
has become a major threat to plant, animal and human life due to their toxic
effect and therefore must be removed from industrial effluent before discharge
(Vinod and Anirudhan, 2001). Deepali and Gangwar (2010) reported that the
soil and ground water samples showed the presence of high level metal
contamination due to the receipt of industrial effluent from tannery industry.
The above results indicate that the raw tannery effluent released into the
atmosphere was found to contain chemicals which were above the permissible
limit prescribed by BIS (2009). The impact of raw effluent on potable water in
the target areas was studied in the next stage.
59
4.1.2. Biochemical profile of the target area water samples

Ground water is the principal source of drinking water in urban and rural
areas of our country. The quality of drinking water in Indian cities has been
detoxified in the recent years mainly due to increase in population and improper
disposal of waste water from industries. These factors have influenced the
surface and ground water (Venkatasubramani et al., 2007). The groundwater in
industrial areas across the country has undergone severe contamination by
industrial waste, effluents and emissions which are discharged indiscriminately
without any regulatory system (Viswanatham et al., 2007). Industrial and
household waste discharged directly or indirectly, through leakages in the
sewage system enters into water sources. The changes in the properties of
water affect the biological lives in that area (Singh, 2006).
The major source of water pollution is industrial and the waste water
generated from various industries which is being discharged into common
drainage. The effluents pollute not only nearby soil but also pollute and alter the
biochemical nature of drinking water. This may be due to the presence of
chemicals in the industrial effluents which pollute and alter the biochemical
nature of drinking water (Bernal et al., 2006).
The organic waste in water may spread pathogenic diseases like
diarrhea and cholera. Along with these diseases, some long term effects can
also occur in some cases (Sathe and Kulkarni, 2001).Tannery effluent when
discharged untreated damages the normal life of receiving stream and if allowed
to percolate into the ground for a prolonged period , seriously affects
the
ground water table of that locality ( Mondel et al., 2003; Prasad, 2007).
Quality of water is an important consideration in any appraisal of salinity
or alkalinity conditions in an irrigated area (Acharya et al., 2008). The water
used for drinking purpose should be free from toxic elements and excessive
amount of minerals that may be harmful to health. Keeping this in focus, to
assess the extent of ground water deterioration, a detailed analysis of ground
60
water quality has been carried out. Biochemical characteristics of ground water
near tannery industry area and water collected from area 15 km away from the
effluent discharge site were analysed and shown in Table 8.
TABLE 8
BIOCHEMICAL PROFILE OF TARGET AREA WATER SAMPLE
S.No
Parameters
Control
water
Target area water

sample
pH
7.21
8.14
Electrical conductivity (mho/cm)
0.65
2.88
Turbidity
0.05
1.28
Total alkalinity
600
914
Total suspended solids
15
418
Total dissolved solids
500
2467
Total hardness
200
830
Calcium hardness
75
454
Magnesium hardness
50
405
10
Fluoride
1.5
0.70
11
Chloride
600
1178
12
Chromium
0.05
10.40
13
Nickel
0.6
3.90
14
Zinc
4.5
7.00
15
Cadmium
1.9
3.40
(mg/l)

Control water- Collected from the area 15 kilometers away from the effluent discharge site
Target area water sample - Water collected within 1 kilometer radius of the effluent discharge
site.
pH and Electrical conductivity (EC)

The pH of the target area water sample was 8.14 and EC was 2.88 in the
present study. The higher pH value indicates the alkaline nature of the effluent.
61
The higher values of EC could be the result of extreme concentrations of soluble

salts from the tannery effluent discharged into the low lands and surface water
bodies of the area. Electrical conductivity and total dissolved solids are
proportional to each other (Gupta et al., 2010). The negative impact of the
effluent on the water quality includes increase in turbidity, colour, nutrient load
and presence of toxic and persistent compounds (Sharief et al., 2005).
Turbidity and Total alkalinity
Turbidity and total alkalinity were found to be increased in the target
area water sample. The total alkalinity of the water is mainly caused by the
contents of calcium, magnesium, sodium, potassium, ammonia and iron,
combined either with carbonates and/or bicarbonates or occasionally by
hydroxide (Jhingran, 2003).
Total suspended solids and Total dissolved solids
Total suspended solids were found to be high in target area water sample
compared to the non contaminated water. Total dissolved solid (TDS) was one
of the often neglected parameter, eventhough it has a tremendous effect on the
overall quality of water. High TDS levels are indicative of the presence of high
levels of both inorganic and organic compounds present in the given water
sample. The total dissolved solids of the target area water sample was high
compared to the BIS value for dissolved solids which is upto 500 mg/l and the
maximum permissible quantity is 2000 mg/l (WHO, 2005). The present study
showed a level of 2467 mg/l of dissolved solids.
The high amount of dissolved solids might be due to the presence
of inorganic salts and small amounts of organic matter dissolved in
water (Lofrano et al., 2008). Jayaseelan et al. (2008) reported that the higher
concentration of total dissolved solids in surface water could be due to the
industrial activities such as tanneries, where huge amount of soluble salts are
used for processing of leather.
62
Total hardness, calcium and magnesium hardness

Hardness is one of the very important properties of ground water
from utility point of view for different purposes. It is a well known fact
that hardness is not caused by a single substance but by a variety of dissolved
polyvalent metallic ions, predominantly calcium and magnesium cations,
although cations like barium, iron, manganese, strontium and zinc also
contribute (Chaudhary et al., 2005). Calcium and magnesium are the principal
ions of hardness.
In the present study, total hardness, calcium and magnesium hardness
were found to be at a higher level in the target area water sample compared to
the control water. Calcium and magnesium causes by far the greatest portion of
the hardness occurring in the natural waters (Acharya et al., 2008). Tanning
process uses these chemicals at greater amounts in liming, pickling and bating
processes which are let out along with the effluent.
Fluoride
Fluoride enters the body through food, water, industrial exposure, drugs
and cosmetics. Presence of fluoride in water may affect the photosynthesis,
respiration and protein synthesis and enzyme activities of higher plants (Sarala
and Rao, 1993). The fluoride content in target area water sample was found to
be lesser than the control water.
Chloride
The chloride content of target area water sample recorded a value of
1178 mg/l which was twofold higher than the chloride content (600mg/l) of the
control water sample. The addition of excess sodium chloride during the tanning
process might be responsible for this.
Chromium, nickel, zinc and cadmium
Chromium, nickel, zinc and cadmium were found to be high in the
target area water sample than the control water. According to World Health
63
Organization (2005), the metals of most immediate concern are aluminum,

chromium, magnesium, manganese, iron, cobalt, copper, nickel, zinc, cadmium,
mercury and lead (Ulmanu et al., 2003) which are discharged into water bodies
that can be toxic to aquatic life and may cause natural water unsuitable as
potable water sources (Cooman et al., 2007). According to Oliveria et al. (2007),
the presence of heavy metals in industrial and urban wastewater is one of the
main causes of water and soil pollution. Accumulation of these elements in
waste water depends on a number of local factors such as the type of industries
in the region, lifestyle and awareness of the impact to the environment by
careless disposal of water (Nordberg et al., 2007).
The results of the study showed that due to unsafe disposal of tannery
waste water on the bare land, the organic and inorganic chemical compounds
present in the effluent have leached and found their way into the ground water.
Hence the potable water in the industrial area was significantly contaminated
with cadmium, chromium, nickel and zinc which were used in the wet finishing
process of tannery process and released along with the effluent.
4.1.3. Physiochemical Characteristics of the target area soil samples
Among various environmental hazards, pollution of soil and water caused
by various effluents has become a serious problem. The tannery industries
discharge large quantities of common salt during the process of tanning.
Deposition of these salts into the soil takes place when the effluent comes in
contact with the soil. Besides chlorides, toxic substances like chromium, sodium
sulphide, sodium carbonate and ammonium sulphate are present in the
discharged effluent which manifolds the soil pollution (Rajan and Arias, 2007).
Heavy metals emitted either from anthropogenic or natural activities can
disperse in the environment and may ultimately get deposited in the soil.
Plants growing in such areas may absorb heavy metals in to their body.
Although metals like iron, molybdenum, manganese, zinc, copper, magnesium,
copper, selenium and nickel have a major role in the growth and development of
plants, they may be toxic beyond a certain level. Long term and indiscriminate
64
application of raw sewage effluent results in the accumulation of these nutrients

into surface and subsurface soils (Vaani and Kumar, 2008).
The dissolved constituents of the industrial waste reacts with clay
complex of soil leading to accumulation of salts and an increase in the amount
of exchangeable sodium and other nutrients. A study on physical and chemical
properties of soil is a basic and practical utility in agriculture. It aims at providing
a tool for proper management of soil (Altieri and Nichollas, 2003).
The tannery waste water being alkaline in nature, leads to the
deterioration of the concrete, metallic pipe through which the effluent passes
and thus causes seepage of the waste water into the land. Free disposal of the
sludge and effluent on the land affects the soil property and fertility of the soil in
the long run.
The selected industry discharges the effluent directly on land. Two soil
types namely red soil and black soil are commonly found in the areas in and
around Dindigul district. Hence, in the present study, the impact of the effluent
on both the soil types were studied and compared with controls of the respective
types.
Physicochemical profile of red soil and black soil
It has to be taken into consideration that sand generally allows the
contaminant to pass into deeper zones. Hence there is a possibility of
contamination of the deeper zones of groundwater in the future if the soil and
groundwater environment are not protected from the tannery wastes. The
presence of heavy metals even in low concentration is an indication of industrial
activity in that area (Zahid et al., 2006). According to Cao and Zhu (1999), red
soil is an important resource for the exploitation and utilization of agriculture and
forestry. Black soil is rich in organic matter in the form of humus. It has large
quantities of nutrients, excellent structure and good water holding capacity,
making it very suitable for agriculture.
65
In the present study, red soil and black soil samples were collected from
the effluent discharge area (Target area soil samples). Red and Black soil
samples collected from the area 15 kms away from the target area served as
control samples.
Table 9 indicates the profile of the selected soil samples.
TABLE 9
PHYSICOCHEMICAL PROFILE OF RED SOIL AND BLACK SOIL
pH
7.5
Target
area red
soil
8.7
EC mho/cm
0.32
0.43
0.19
0.87
Sodium
122
52
32.0
24.82
Nitrogen
95
98
66
73
Phosphorous
8.57
14.06
4.25
8.21
Potassium
474
650
149
378
Calcium
1.25
2.38
0.45
0.63
175
140
164
138
Copper
15.86
62
4.86
19.64
Iron
63.0
86.0
20.9
52.9
Chromium
22.01
93
15.52
48.72
2.0
8.7
2.6
9.6
18.22
29.05
8.72
11.83
1.5
7.4
1.8
9.2
Parameters
Magnesium
Red soil
(control)
mg/kg
Nickel
Zinc
Cadmium
Black soil
(control)
Target area
black soil
7.15
8.8

Control soil- Soil collected from 15 kilometers away from the effluent discharge site
Target area soil sample - Soil collected within one kilometer radius of the effluent discharge
site.
Soil pH is one of the most influential parameters controlling the

conversion of metals from immobile solid phase forms to more mobile form
(Abou et al., 2008). pH of the target area black soil was found to be 8.8 and that
66
of red soil was found to be 8.7. Zahid et al., (2006) reported that the pH varies
between 7.5 and 8.5 in the top soil, where carbonates generally precipitate
and many trace metals co precipitate with them and make the soil alkaline.
Sinha et al.(2006) suggested that soil becomes alkaline due to the alkalinity of
the tannery effluent discharged.
Electrical conductivity of the target area red soil (0.43 mho/cm) was
greater compared to the control red soil (0.32 mho/cm). The conductivity of the
target area black soil was found to be 0.87 which was higher compared to that
of control black soil (0.19). High conductivity of the soil indicates the presence of
higher levels of anions and cations in the soil. Soil discharged with effluents
from cotton ginning mills and paper mills showed higher electrical conductivity
(Medhi, 2005). The addition of tannery effluent to the soil affects the physical
properties of the soil. The effluent is rich in salts, particularly sodium chloride,
which on continuous irrigation increased the concentration in soil and reflected
in increased electrical conductivity (Thangavel et al., 2003).
Levels of sodium in the target area red soil were found to be 52 mg/kg
which was less than the control red soil (122 mg/kg). 24.82 mg/kg of sodium
was found to be present in target area black soil whereas 32 mg/kg of sodium
was found in control black soil (Figure 2). Sodium content in the red soil was
higher than that of the black soil. Krishna and Govil (2008) reported that the
level of cations (sodium and potassium) in the soil irrigated with tannery waste
water varied differently from control sites.
Generally nitrogen is the growth limiting nutrient, which is needed in the
highest concentration. Nitrogen content in the target area black soil (73 mg/kg)
was found to be higher than that of the control black soil (66 mg/kg). The target
area red soil contained 98 mg/kg nitrogen (Figure 2) which was on par with the
control red soil (95 mg/kg). According to Kabdali et al. (2003), the high nitrogen
content in the target area soil might favour plant growth.
67
FIGURE 2
SODIUM AND NITROGEN CONTENT IN THE TARGET AREA SOILS
140
CR
TR
120
CB
TB
mg/kg
100
80
60
40
20
0
Sodium
Nitrogen
CR - Control red soil
TR - Target area red soil
CB - Control black soil
TB - Target area black soil
The phosphorus content of the target area red soil was found to be
14.06 mg/kg, which was greater than the control red soil (8.57 mg/kg).
Phosphorus content in the target area black soil was found to be 8.21 mg/kg
while that of control soil was found to be 4.25 mg/kg. The results of the study
conducted by Chonkar et al., (2003) revealed that the phosphorus content in soil
increased significantly due to application of industrial sludge.
Potassium levels in the target area red soil (650 mg/kg) were found to
be higher than the control red soil (474 mg/kg). Target area black soil was found
to contain 378 mg/kg of potassium and control black soil without effluent was
found to contain 149 mg/kg (Figure 3). Nitrogen, phosphorus and potassium
contents in both the soils were found to be higher in target area soil compared
to that of the control soil.
Nitrogen, phosphorus, potassium and calcium contents were increased in
soils after disposal of sewage wastes (Girisha et al., 2006). Soil analysis in the
68
study done by Sheela and Peethambaram (2007) revealed that NPK content of
the soil was slightly increased by effluent treatment as the nutrients nitrogen,
phosphorus and potassium present in diluted effluent played a role in promoting
plant growth at lower concentration. Irrigation with sewage and pulp paper cult
effluent was reported to enrich the soil, mainly with respect to nitrogen,
phosphorus and potassium, and enhanced the crop yields considerably (Nan
and Chung, 2001).
FIGURE 3
POTASSIUM AND MAGNESIUM CONTENT IN THE
TARGET AREA SOILS
700
600
CR
TR
CB
TB
mg/kg
500
400
300
200
100
0
Potassium
Magnesium
Calcium levels of red soil in target area with tannery effluent were higher
than the control red soil (2.38 mg/kg and 1.25 mg/kg respectively). Calcium
content of the target area black soil and control black soil were 0.63 mg/kg and
0.45 mg/kg respectively. Red soil had a higher concentration of calcium
compared to that of black soil.
The red soil collected from the target area was found to contain a low
level of magnesium (140 mg/kg) compared to the control red soil which had
175 mg/kg of magnesium (Figure 3). Magnesium content in the black soil of
69
target area was lesser than that of the control black soil (138 mg/kg and 164
mg/kg respectively). Target area red soil was found to contain a higher level of
magnesium compared to the target area black soil.
A threefold increase in copper content was recorded in target area red
soil compared to that of the control red soil. The content of copper in target area
black soil was found to be 19.62 mg/kg while 4.86 mg/kg of copper was found to
be present in control black soil (Figure 4). Though copper is a micronutrient of
prime importance in agricultural production, it may cause environmental problem
when accumulated in soils. Adsorption of copper depends upon soil properties
like pH, organic matter, clay and cation exchange capacity (Anuradha, 2005).
Copper is an essential element and good for health in very small quantities but
at excessive dose it is toxic.
FIGURE 4
CHROMIUM, ZINC AND COPPER CONTENT IN THE
TARGET AREA SOILS
100
CR
TR
90
CB
TB
80
mg/kg
70
60
50
40
30
20
10
0
Chromium
Zinc
Copper
Iron content was higher in target area red soil (86.0 mg/kg) than control
red soil. In black soil 52.9 mg/kg of iron was present in target area sample which
70
was higher than the control black soil.
Increased amount of iron may be
contributed by the weathering of rocks and also by the discharge of effluent and
other wastes on surface that percolated into the ground water (Jain et al., 2000).
Higher values of iron were noted in tannery effluent contaminated soil
(Alverz-Bernal et al., 2006). As suggested by Xiong et al. (2001) untreated
industrial effluent contains higher amounts of cadmium, lead, zinc, copper,
manganese and iron which would have enhanced the concentration of heavy
metals in irrigated soil. From the composition of the heavy metals in the soils,
it was obvious that tannery industries were responsible for not only the increase
in chromium content in the soil inherent to the tanning process but also
cause an increase in iron, zinc, manganese, copper and sulphate contents
(Zahid et al., 2006).
Rajkumar et al. (2005) observed that chromium is a transition metal that
is discharged into the environment through the disposal of wastes from
industries like leather tanning and metallurgical, leading to contamination of soil.
Chromium is the main tanning agent and most hazardous chemical used in
chrome tanning process. The excessive use of this chemical leads to higher
concentration in the effluent (Bhalli and Khan, 2006). Chromium levels in the
target area red soil was found to be increased three fold than the control red soil
(93 mg/kg and 22.01 mg/kg respectively). The target area black soil was found
to contain 48.72 mg/kg of chromium while that of control black soil was
15.52 mg/kg (Fig 4). It is the major chemical present in the effluent, which, when
released into the soil, percolates the layers of soil.
Krishna and Govil (2008) reported that the level of chromium was found
to be high in soils receiving treated tannery waste water for irrigation than
control soil. Inadequate disposal of waste containing chromium at industrial site
had contaminated both the ground and water. High chromium levels have been
well documented to have negative impact on plant growth.
The results show that the target area red soil had 8.7mg/kg and control
red soil had 2.0 mg/kg of Nickel. Nickel level in target area black soil was found
71
to be 9.6 mg/kg compared to that of control black soil which was 2.6 mg/kg
(Figure 5).
Presence of chromium and nickel in soil disturbs the pattern of
nutrient uptake by increasing or decreasing the nutrient content in plants

due to the heavy metal and nutrient metal interactions (Zupansic et al., 2004;
Harikrishnan and Kumar, 2009).
FIGURE 5
NICKEL AND CADMIUM CONTENT IN THE TARGET
AREA SOILS
12
CR
TR
CB
TB
10
mg/kg
8
6
4
2
0
Nickel

Cadmium

The metals, zinc and cadmium, were found to be 29.05 mg/kg and
7.4 mg/kg respectively in target area red soil which were higher compared to the
control red soil (18.22 mg/kg and 1.5 mg/kg respectively). Zinc and cadmium
content in the target area black soil were found to be 11.83 mg/kg and
9.2 mg/kg respectively while those of control black soil were 8.72 mg/kg and
1.8 mg/kg respectively (Figure 4 and 5). Higher amounts of heavy metals like
copper, zinc, iron and manganese were recorded in irrigated soil near industrial
complex as reported by Barman et al.(2001).
Excessive accumulation of heavy metals such as cadmium, lead,
chromium and nickel in the soil due to effluent discharge and the resultant
phytotoxicity was reported by Peralta et al. (2001) and Tsakou et al.(2001).
72
Cadmium dispersed in the environment persists in soils and sediments

for decades and taken up by plants which accumulates in the biosphere
(Bernard, 2008).
According to Sinha et al. (2006), chromium and other metals were found
to be high in effluent contaminated soils. Significant increases in soil metal
content were found in areas of high industrial activity where accumulation may
be several times higher than that of the average content in non contaminated
areas (Krishna and Govil, 2008).
From the results of the study it was observed that control red soil was
found to be rich in minerals and nutrients compared to the control black soil. The
contents of sodium, nitrogen, phosphorus, potassium, calcium and magnesium
were found to be at a higher level in target area red soil compared to the target
area black soil.
PHASE II
4.2 Growth studies of selected plants using diluted tannery effluent
The use of plants to improve water quality in municipal and more recently
industrial waste water treatment system is of great use and an emerging
technology of phytoremediation (Suseela et al., 2002). Girisha et al. (2006) in
their study acclaimed that tannery and textile industrial waste water contains
appreciable amount of plant nutrients such as nitrogen, phosphorus, potassium,
calcium, magnesium and sulphur. These nutrients could be used for plant
growth after proper treatment.
Direct discharge of effluents changes the physico chemical and biological
characteristics of soils and was responsible for the reduction in the rate of
germination of seeds but studies have proved that properly diluted effluents can
be used for irrigation (Sheela and Soumya, 2004).
Favorable effect of diluted effluents on seedling growth have been
investigated and well documented (Chandra et al., 2009; Shreshtha and
Niroula, 2003).
73
Plants Vigna radiata and Vigna mungo belong to the family leguminosae.
They have been recognized for their economic value and are rich in protein,
calcium, phosphorus and vitamins (Simanova et al., 2007). These leguminous
plants have a property of impermeability of seed coat and also reported to show
resistance against salinity. Since it has been well documented that raw effluent
discharged from the tannery industry has a detrimental effect on the plant
growth, in the present study, various dilutions of the effluent were used for the
measurement of growth of the selected plants Vigna radiata and Vigna mungo.
Nutrients added through the effluent were retained to some extent; at the same
time the high salt concentration of the effluent was reduced by dilution. Several
growth parameters such as percentage of germination, seedling survival,
seedling height, yield parameters and biochemical parameters have been taken
as criteria to assess plant responses to a specific pollutant.
In the present study, plants Vigna radiata and Vigna mungo were grown
in effluent contaminated red soil and black soil. The growth was continued with
25% and 50% tannery effluent. Plants were grown in red soil (CR), red soil +
25% effluent (T1R), red soil+50% effluent (T2R), black soil (C2B), black soil +
25% effluent (T1B), black soil + 50% effluent (T2B) for 90 days till the seeds
were produced. Germination percentage and vigour index were observed on
8th day after sowing. Selected biometric observations (root length, shoot length,
fresh weight, dry weight, number of leaves and number of roots), selected
biochemical parameters such as chlorophyll, carotenoid, carbohydrate and
protein in the leaf samples, total phenol, carotenoid, carbohydrate and protein
contents in the seed samples were analysed. Leaves and fresh seeds of all the
plants were analysed for their enzymic and non enzymic antioxidant status.
Metals like chromium, nickel, zinc and cadmium were analysed in roots,
shoots, leaves and grains of both the plants grown in red and black soil using
both the effluent concentrations. Histochemical staining of root samples were
observed for the accumulation of metals and yield parameters such as number
of nodules, flowering time, pod weight, pod length, number of pods per plant,
74
number of seeds per plant, number of seeds per pod, 100 seed weight and total
seed weight per plant were studied.
4.2.1 Biometric observations of the plant samples
Plant growth can be best monitored by observing the biometric
observations of the plants. Tannery effluent has a higher concentration of total
nitrogen, sulphide, dissolved and suspended solids and due to excess of these
nutrients plant growth is inhibited as it affects the water absorption and other
metabolic processes of the plant (Sheela and Peethambaram, 2007). Panda
and Choudry (2005) and Yongpisanphop et al. (2005) observed that raw effluent
irrigation adversely affects the plant growth and development but use of diluted
effluent would enhance plant growth.
4.2.1.1 Germination percentage and vigour index
Plant growth and development are essential processes of life and
propagation of the species. They are continuous and mainly depend on external
resource present in soil and air. Growth is chiefly expressed as a function of
genotype and environment, which consists of external and internal growth
factors. Presence of contaminants in the external environment leads to change
in the growth and development pattern of the plant.
Among the growth process, seed germination and seedling growth have
been considered critical for raising a successful agricultural crop. The process of
germination and growth of young seedlings are susceptible to toxic materials
in water. The ability of a crop to germinate and establish under stress by
environmental contaminants is an early indicator of tolerance of the plant. Seed
germination was the first physiological process affected by the heavy metals
when present in soil and water (Peralta et al., 2001).
Table 10 and Figure 6 and 7 show the percent germination and vigour
index of the plants Vigna radiata and Vigna mungo grown in red soil and black
soil using 25% and 50% tannery effluent. These parameters were observed on
the 8th day after sowing.
75
TABLE 10
GERMINATION PERCENTAGE AND VIGOUR INDEX OF Vigna radiata AND
Vigna mungo GROWN IN RED SOIL AND BLACK SOIL USING
DILUTED TANNERY EFFLUENT
Germination percentage
Soil
Groups
Vigour index
CR
Vigna
radiata
98.0 1.63
Vigna
mungo
96.0 3.27
Vigna
radiata
392 2.43
Vigna
mungo
364 5.20
T1R
94.0 3.01
90.7 6.94
329 1.58
300 4.90
T2R
91.0 3.15
86.0 4.20
273 2.45
249 2.05
CB
96.0 4.90
94.0 3.17
393 2.42
366 7.06
T1B
93.0 2.35
91.0 4.18
344 3.24
327 2.43
T2B
CD ( 0.05)
81.0 3.64
84.0 3.16
259 3.30
260 4.05
Red
soil
Black
soil
8.11
6.36
Values are mean SD of triplicates

CR-Control red soil, T1R-Red soil with 25% effluent, T2R-Red soil with 50% effluent,
CB-Control black soil, T1B-Black soil with 25% effluent, T2B- Black soil with 50% effluent.
FIGURE 6
GERMINATION PERCENTAGE OF Vigna radiata AND
Vigna mungo
Germination percentage
100
90
80
70
60
50
40
30
20
10
0
Vigna radiata
CR
T1R
Vigna mungo
T2R
CB
T1B
T2B
CR-Control red soil, T1R-Red soil with 25% effluent, T2R-Red soil with 50% effluent,
CB-Control black soil, T1B-Black soil with 25% effluent, T2B- Black soil with 50%
effluent.
76
FIGURE 7
VIGOUR INDEX OF Vigna radiata AND Vigna mungo
450
400
Vigour index
350
300
250
200
150
100
50
0
Vigna radiata
CR
T1R
Vigna mungo
T2R
CB
T1B
T2B
Seeds of Vigna mungo grown with 50% effluent showed a significant

reduction in germination compared to the control plants. Both the plants grown
in black soil with 50% tannery effluent showed a significant reduction in
germination percentage compared to the control plants of black soil.
A significant reduction (p< 0.05) in vigour index was observed in both the
plants grown in both the soil types using 25% and 50% effluent compared to the
control plants.
According to Malla and Mohanty (2005), there was significant decrease in
percentage germination, root length and shoot length with increase in the
effluent concentration. In their work with mung seeds, they reported that
percentage germination and the concentration of effluent treatment were
negatively correlated. Pandey et al. (2008) reported that supply of the undiluted
distillery effluent produced significant inhibition in seed germination
4.2.1.2 Root length
Table 11 shows the root length of the plants Vigna radiata and Vigna
mungo grown in red and black soils using 25% and 50% tannery effluent.
77
2.90 0.12
4.20 0.11
CB
T2B
3.10 0.16
T2R
3.40 0.33
3.40 0.3
T1R
T1B
4.00 1.63
30
CR
Groups
2.92
4.70 0.20
5.70 0.10
6.00 1.53
3.00 0.82
4.67 0.94
6.00 3.27
60
6.80 0.19
7.80 0.17
8.40 0.13
6.00 2.35
7.00 2.20
9.00 2.45
3.27 0.39
4.70 0.21
5.30 0.24
4.50 0.41
5.00 2.38
6.00 2.43
Days after sowing (DAS)

90
30
3.43
5.80 0.18
6.80 0.13
7.80 0.14
5.00 3.27
6.20 0.16
7.00 2.30
60
Vigna mungo
7.30 0.24
8.60 0.21
8.90 0.08
7.50 0.41
8.00 1.63
10.00 4.08
90
78
78
CR- Control red soil, T1R- Red soil with 25% effluent, T2R- Red soil with 50% effluent, CB- Control black soil, T1B- Black soil with 25% effluent,
T2B- Black soil with 50% effluent.
CD ( 0.05)
Black
soil
Red soil
Soil
Vigna radiata
ROOT LENGTH (cm) OF Vigna radiata AND Vigna mungo GROWN IN RED AND BLACK SOILS USING DILUTED
TANNERY EFFLUENT ON 30, 60 AND 90 DAYS AFTER SOWING
TABLE 11
Plant biomass, root length and shoot length are used as indices of growth
performance. Tolerance to toxic elements and biotic stress depends on well
branched and extensive root systems. In our study, Vigna radiata and Vigna
mungo highlighted significant differences in their responses to various
concentrations of tannery effluent.
It was observed that both the plants recorded no significant difference in
the root length when grown with 25% effluent in both the soils. A significant
reduction in root length was observed on 90th day only when Vigna radiata was
grown with 50% effluent. The reduction might be due to the metals present in
the tannery effluent.
According to Pandey et al. (2008) supply of untreated effluent produced
significant inhibition in seed germination and seedling growth parameters in both
maize and rice. According to them a significant inhibition in root length was
observed with 50% effluent.
Studies on Allicum cepa (Palacio et al., 2005), Zea mays (Akbar et al.,
2009) and Arachis hypogea (Nagajyoti et al., 2009) showed significant decrease
in root length with higher chromium concentration in soil. The reduction in root
growth could be due to the direct contact of seedling roots with pollutants
causing a collapse and subsequent inability of the root to absorb water.
4.2.1.3. Shoot length
Shoot length is considered as an important morphological parameter
related to growth and development of the whole plant.
Table 12 shows the shoot length of the plants Vigna radiata and Vigna
mungo grown in red soil and black soil using 25% and 50% tannery effluent on
30th, 60th and 90th days after sowing.
Both the plants recorded significant reduction in shoot length when grown
with 50% effluent in red soil. In black soil Vigna mungo recorded significant
reduction on the 60th day of growth.
79
16.0 2.19
T2B
21.0 2.34
CB
17.0 2.30
16.0 2.47
T2R
T1B
20.0 2.32
23.0 2.45
30
T1R
CR
Groups
5.36
34.0 3.32
35.0 4.08
38.0 2.40
35.33 2.87
37.0 3.17
40.0 2.42
60
53.0 2.65
54.0 3.06
57.0 2.42
58.0 1.73
63.0 2.35
68.0 1.63
90
16.0 1.74
17.0 1.45
20.0 3.24
14.0 3.47
19.0 0.72
22.0 1.58
30
4.95
32.0 1.62
36.0 2.27
37.0 2.25
32.0 1.43
34.0 3.27
37.0 2.41
60
Vigna mungo
51.0 1.60
52.0 1.43
55.0 2.31
47.0 2.15
49.0 0.82
55.0 4.08
90
80
80
CD ( 0.05)
Black
soil
Red soil
Soil
Vigna radiata
SHOOT LENGTH (cm) OF Vigna radiata AND Vigna mungo GROWN IN RED AND BLACK SOILS USING DILUTED
TABLE 12
Purohit et al. (2003) reported that the shoot length of tomato plant
decreased when the concentration of the tannery effluent was increased. Babu
and Vishnuvardhan (2006) reported that the shoot length and root length of
Vigna mungo was retarded and inhibited at higher effluent concentration.
According to Rajula and Padmadevi (2000), the germination percentage
and morphological characters like shoot length and root length decreased
gradually with increase in effluent concentration. A gradual decrease in
germination percentage, root length, shoot length, fresh weight and dry weight
of black gram seedlings with increase in chromium concentrations was
observed by Chidambaram et al., (2009).
4.2.1.4 Fresh weight
Table 13 shows the fresh weight of Vigna radiata and Vigna mungo
plants grown in red soil and black soil using 25% and 50% tannery effluent.
Vigna radiata grown with black soil using 50% effluent showed significant
reduction of fresh weight on 90th day compared to that grown with red soil.
Vigna mungo recorded significant reduction (p<0.05) of fresh weight on 90th day
with 50% effluent using red soil compared to that with black soil.
Chidarambam et al., (2009) in their work on black gram (Vigna mungo)
reported a gradual decrease of root length, shoot length, fresh weight and dry
weight with increase in chromium concentrations.
Decrease in the fresh weight may be the outcome of a decreased water
uptake or enhanced water loss, both of which may occur following membrane
damage since plant cell membranes are generally considered as the primary
sites of metal injury (Diwan et al., 2010b).
81
16.0 0.72
T2B
17.0 2.34
CB
16.0 1.43
16.0 3.23
T2R
T1B
19.0 0.91
21.0 3.27
30
T1R
CR
Groups
4.76
25.0 4.18
26.0 3.43
27.0 2.41
26.0 2.11
28.0 1.53
29.0 0.82
60
53.0 2.35
56.0 3.04
58.0 1.73
59.0 0.82
61.0 2.25
63.0 2.45
90
15.0 2.49
15.0 4.08
16.0 3.19
17.0 2.41
18.0 2.43
18.0 1.63
30
6.53
23.0 2.61
24.0 3.17
25.0 3.56
22.7 3.72
25.0 4.08
29.0 0.92
60
Vigna mungo
49.0 0.22
52.0 2.38
53.0 2.02
51.0 3.37
53.0 1.23
84.0 3.65
90
82
82
CD ( 0.05)
Black
soil
Red soil
Soil
Vigna radiata
FRESH WEIGHT (g) OF Vigna radiata AND Vigna mungo GROWN IN RED AND BLACK SOILS USING DILUTED
TABLE 13
4.2.1.5. Dry weight

Table 14 shows the dry weight of the plants Vigna radiata (Figure14)
and Vigna mungo (Figure 15) grown in red soil and black soil using 25% and
50% tannery effluent on 30th, 60th and 90th days after sowing.
Vigna radiata plants grown in red soil did not show any differnece in their
dry weights on comparision with control plants. While on growth with black soil
no significance difference was obtained on 30th and 60th day. But on 90th day a
significant reduction was observed in 50% effluent.
Vigna mungo plants grown in red soil was found to have no significant
difference in dry weight when grown in 25% effluent. But with 50% effluent a
signficant difference was noticed on 30th day plant. Plants grown with black soil
showed a significant reduction on 90th day.
This indicated that plants showed a better growth with 25% effluent which
is more diluted compared to 50% effluent. In a study conducted by Diwan et al.,
(2010), significant reduction in dry weight was observed due to increased
effluent concentration.
4.2.1.6. Number of leaves and Number of roots
Table 15 and 16 show the number of leaves and roots of the plants Vigna
radiata and Vigna mungo grown in red soil and black soil using 25% and 50%
tannery effluent on 30th, 60th and 90th day after sowing.
Vigna raidiata and Vigna mungo plants grown in 25% and 50% effluent
had no significant difference in their number of leaves when grown in red soil
and black soil.
No. of roots of Vigna radiata and Vigna mungo plants showed a gradual
increase from 30th to 90th day of growth. Plants grown in red soil and black soil
followed the same trend and found to be on par with the control plants. Similarly
with effect of different dilutions of the effluent (25% and 50%) was not found to
be significant on the number of roots.
83
1.60 0.21
T2B
1.80 0.14
CB
1.70 0.34
1.70 0.74
T2R
T1B
1.90 0.08
2.0 0.41
30
T1R
CR
Groups
0.47
2.20 0.24
2.20 0.12
2.50 0.44
2.13 0.29
2.30 0.24
2.70 0.13
60
2.91 0.01
3.21 0.04
3.57 0.06
3.01 0.03
3.16 0.05
3.46 0.03
1.70 0.08
1.50 0.37
1.80 0.26
1.50 0.31
2.03 0.69
2.60 0.33

90
30
0.60
2.10 0.16
2.50 0.45
2.60 0.23
2.0 0.16
2.30 0.24
2.60 0.28
60
Vigna mungo
3.20 0.13
3.80 0.19
4.0 0.42
3.71 0.23
3.86 0.02
4.01 0.01
90
84
84
CD ( 0.05)
Black
soil
Red soil
Soil
Vigna radiata
DRY WEIGHT (g) OF Vigna radiata AND Vigna mungo GROWN IN RED AND BLACK SOILS USING DILUTED
TABLE 14
10.0 4.18
T2B
13.0 2.65
CB
11.0 1.53
11.33 2.87
T2R
T1B
12.0 1.61
13.0 2.45
30
T1R
CR
Groups
5.64
19.0 1.23
20.0 4.06
23.0 2.38
20.0 4.02
21.0 1.58
24.0 3.27
60
29.0 0.82
32.0 0.32
34.0 3.17
32.0 1.43
34.0 3.27
35.0 4.08
6.0 3.46
9.0 0.62
10.0 2.45
7.0 2.25
9.0 0.72
10.0 4.05

90
30
4.23
17.0 2.32
20.0 1.51
21.0 1.69
18.0 1.43
19.0 0.61
21.0 0.82
60
Vigna mungo
29.0 0.81
27.0 0.80
31.0 0.34
29.0 2.51
31.0 3.11
32.0 1.63
90
85
85
CD ( 0.05)
Black
soil
Red soil
Soil
Vigna radiata
NUMBER OF LEAVES OF Vigna radiata AND Vigna mungo GROWN IN RED AND BLACK SOILS USING
DILUTED TANNERY EFFLUENT ON 30, 60 AND 90 DAYS AFTER SOWING
TABLE 15
6.0 1.68
T2B
9.0 1.43
CB
8.0 1.67
7.0 2.43
T2R
T1B
8.0 1.66
9.0 0.82
30
T1R
CR
Groups
4.05
12.0 1.61
11.0 2.49
13.0 2.65
9.0 0.52
10.0 1 .18
12.0 1.63
60
10.0 0.80
12.0 1.69
13.0 0.42
13.0 2.44
12.0 0.83
14.0 1.27
7.0 2.47
8.0 2.41
9.33 2.05
8.33 1.35
9.33 1.25
10.0 1.08

90
30
5.29
11.0 0.80
10.0 4.07
12.0 1.33
11.0 0.72
10.0 4.04
13.0 1.45
60
Vigna mungo
12.0 1.62
11.0 1.65
13.0 2.75
14.0 3.07
12.0 1.62
15.0 1.06
90
86
86
CD ( 0.05)
Black
soil
Red
soil
Soil
Vigna radiata
NUMBER OF ROOTS OF Vigna radiata AND Vigna mungo GROWN IN RED AND BLACK SOILS USING DILUTED
TABLE 16
4.2.2. Biochemical parameters

Morphological or physiological changes in plants will not be visible unless
there is certain metabolic change in cellular activities such as in photosynthesis.
Essential heavy metals like iron, manganese, zinc and copper for all higher
plants are absorbed and accumulated in plant tissues based on their solubility,
concentration and availability of different ions in the soils. Excess accumulation
of heavy metals causes either deficiency or enrichments of other essential
micronutrients which will a have direct effect on the concentration of chlorophyll
(Sharma and Chettri, 2008).
The toxic chemicals and pollutants especially heavy metals present in the
tannery effluent may affect the biochemical characteristics such as chlorophyll,
carotenoids, carbohydrate, protein and enzymes such as glucose 6 phosphate
dehydrogenase, peroxidase and superoxide dismutase (Singh and Tiwari, 2003;
Sharma and Agrawal, 2005). The subsequent biochemical changes of the plant
samples due to waste water irrigation were studied by Gupta et al., (2010).
Hence in the present study an attempt was made to study the changes
in biochemical parameters after irrigating the selected plants with various
concentrations of tannery effluent. Biochemical parameters were analysed in
roots, shoots, leaves and seeds of both the plants grown in 25% and 50%
tannery effluent.
4.2.2.1. Biochemical parameters in the leaf samples
Biochemical parameters such as chlorophyll, carotenoid, carbohydrate
and protein were analysed in the leaves of both the plants grown with 25% and
50% tannery effluent. Estimation was done on the leaves of plants collected on
30, 60 and 90 days after sowing.
87
0.29 0.02
T4
0.40 0.04
C2
0.39 0.03
0.32 0.03
T2
T3
0.41 0.04
SD of triplicates
0.004
0.20 0.04
0.29 0.05
0.31 0.03
0.21 0.02
0.31 0.03
60
0.371 0.03
0.09 0.02
0.16 0.04
0.20 0.03
0.10 0.02
0.19 0.04
0.18 0.01
0.25 0.02
0.42 0.03
0.46 0.04
0.57 0.05

90
30
0.22 0.03
0.71 0.02
0.002
0.14 0.04
0.22 0.05
0.35 0.02
0.36 0.03
0.46 0.04
60
0.63 0.05
Vigna mungo
0.11 0.01
0.17 0.02
0.19 0.01
0.27 0.02
0.34 0.03
90
0.49 0.04
88
88
CR- Control with red soil, T1R- Red soil with 25% effluent, T2R- Red soil with 50% effluent, CB- Control black soil, T1B- Black soil with 25%
effluent, T2B- Black soil with 50% effluent
Values are mean
30
0.48 0.04
T1
C1
Groups
CD (0.05)
Black
soil
Red soil
Soil
Vigna radiata
CHLOROPHYLL CONTENT (mg/g) IN THE LEAVES OF Vigna radiata And Vigna mungo GROWN IN RED AND
BLACK SOIL Using DILUTED TANNERY EFFLUENT
TABLE 17
a) Chlorophyll
Chlorophylls are the most important pigments in plants because of their
photosynthetic process. A quantitative analysis of these pigments is important in
elucidating the photosynthetic productivity of plants. Synthesis of chlorophyll
is controlled by the interaction of a number of environmental factors such as
light, temperature, oxygen, moisture content, metallic ions and nutrients.
Table 17 shows the chlorophyll content in the leaves of Vigna radiata
(Figure 8) and Vigna mungo (Figure 9) grown in red and black soil using 25%
and 50% tannery effluent.
FIGURE 8
Chlorophyll content (mg/g)
CHLOROPHYLL CONTENT IN Vigna radiata

0.6
CR
T1R
T2R
0.5
CB
T1B
T2B
0.4
0.3
0.2
0.1
0
30 DAS
60 DAS
90 DAS
Chlorophyll content in the leaves of both the plants in both the soils using
25% and 50% tannery effluent were found to be significantly reduced than that
of the plants grown without effluent. A decrease in the chlorophyll content
suggests pollution injury.
Mishra et al. (2008), in their study demonstrated that the levels of
chlorophyll and protein decreased in Eichornea crassipies when the metal levels
increased. According to Sharma and Chettri (2008) excess heavy metals
89
accumulated in different plant tissues may influence the disturbances in plant

nutrition. Thus, excess accumulation of heavy metals causes either deficiency
or enrichment of other nutrients which has a direct effect on chlorophyll.
Magnesium of chlorophyll molecule is substituted by heavy metals (copper,
cadmium, nickel, lead) resulting in heavy metal substituted chlorophylls.
FIGURE 9
CHLOROPHYLL CONTENT IN Vigna mungo
0.8
Chlorophyll content (mg/g)
0.7
CR
T1R
T2R
CB
T1B
T2B
0.6
0.5
0.4
0.3
0.2
0.1
0
30 DAS
60 DAS
90 DAS
Accumulation of copper, zinc and cadmium would have inhibited the

chlorophyll biosynthesis due to prevention of photoreactive protochlorophyll
reductase complex formation and amino levulinic acid synthase as opined
by Singh et al. (2003). Reduction in chlorophyll content may be attributed to
impaired uptake of essential elements, damage of photosynthetic apparatus or
due to chlorophyll degradation by increased chlorophyllase activity (Sharma and
Dubey, 2005). The chemicals released from tannery effluent might be the
reason for the reduction in chlorophyll content in the experimental plants.
b) Carotenoid
Table 18 shows the carotenoid content in the leaves of the plants Vigna
radiata and Vigna mungo grown in red soil and black soil grown using 25% and
50% effluent.
90
0.03 0.02
T4
0.04 0.02
C2
0.04 0.05
0.03 0.01
T2
T3
0.04 0.02
0.04 0.01
30
T1
C1
Groups
0.06
0.2 0.02
0.8 0.01
1.10 0.1
0.11 0.01
0.12 0.03
0.12 0.02
60
1.01 0.4
1.02 0.3
1.02 0.3
1.02 0.2
1.03 0.3
1.03 0.1
90
0.04 0.05
0.05 0.02
0.05 0.02
0.05 0.02
0.06 0.5
0.07 0.05
30
0.01
0.1 0.01
0.2 0.02
1.11 0.1
0.10 0.1
0.12 0.02
0.20 0.02
60
Vigna mungo
1.0 0.2
1.1 0.3
1.02 0.5
0.08 0.5
1.02 0.1
1.03 0.1
90
91
91
CD (0.05)
Black
soil
Red soil
Soil
Vigna radiata
CAROTENOID CONTENT (mg/g) IN THE LEAVES OF Vigna radiata AND Vigna mungo GROWN IN
RED AND BLACK SOIL USING DILUTED TANNERY EFFLUENT
TABLE 18
Carotenoid content in the leaves of Vigna radiata showed significant

reduction with black soil grown in 50% tannery effluent only on 60th day of
growth whereas Vigna mungo recorded significant reduction of carotenoid
content in both the soils grown with 25% and 50% effluents from 60th day to 90th
day.
Earlier reports suggested that effects of heavy metals on carotenoid
content were plant and metal specific. Chromium induced degradation of
carotenoid has been reported in V.spiralis by Vajpayee et al. (2001).
c) Carbohydrate
Table 19 shows the carbohydrate content in the leaves of Vigna radiata
(Figure 10) and Vigna mungo (Figure 11) plants grown in red soil and black soil
using 25% and 50% tannery effluent.
The carbohydrate content in the leaves of Vigna radiata and Vigna
mungo (control) plants increased with increase in time of growth and showed a
maximum in the 90th day plant. The carbohydrate content in the leaves of the
plants grown with 25% effluent showed a value of 4.18 and with 50% effluent
it was 3.16 mg/g. Similarly the carbohydrate content of the leaves of Vigna
radiata plants grown in black soil with 25% and 50% effluent were 3.78 and
2.03 mg/g respectively on 30th day. A significant decrease was observed in the
carbohydrate content of the leaves of T1R, T2R, T1B and T2B plants compared
to control plants.
When the concentration of pollutants in the effluent exceeds the
detoxifying capacity of the tissue through their normal metabolism, there is a
decrease in the biochemical parameters such as chlorophyll, protein, amino
acid, carbohydrate and nucleic acid (Hamid and Jawaid, 2009; Adekunle et al.,
2010).
Changes in pigment concentration by effluent treatment affected the
carbohydrate content in a study conducted by Malla and Mohanty (2005) which
supports our observation.
92
2.03 0.4
T2B
4.18 0.42
CB
3.78 0.5
3.16 0.36
T2R
T1B
4.18 0.55
4.86 0.54
30
T1R
CR
Groups
0.01
2.95 0.35
4.07 0.25
4.90 0.56
3.70 0.65
4.92 0.28
5.32 0.25
60
3.27 0.28
4.97 0.88
5.15 0.95
4.41 0.46
5.70 0.33
6.21 0.26
2.93 0.82
3.71 0.73
3.90 0.45
3.45 0.48
4.82 0.69
4.96 0.85

90
30
0.01
4.02 0.29
4.25 0.85
4.44 0.34
4.84 0.34
5.36 0.62
5.73 0.68
60
Vigna mungo
5.35 0.38
5.65 0.95
5.87 97
5.14 0.48
5.86 0.95
6.81 0.86
90
93
93
CD (0.05)
Black
soil
Red soil
Soil
Vigna radiata
CARBOHYDRATE CONTENT (mg/g) IN THE LEAVES OF Vigna radiata AND Vigna MUNGO GROWN IN
RED AND BLACK SOIL USING DILUTED TANNERY EFFLUENT
TABLE 19
FIGURE 10
CARBOHYDRATE CONTENT IN THE LEAVES
OF Vigna radiata
7
Carbohydrate content (mg/g)
CR
T1R
T2R
CB
T1B
T2B
5
4
3
2
1
0
30 DAS
60 DAS
90 DAS
FIGURE 11
CARBOHYDRATE CONTENT IN THE LEAVES
OF Vigna mungo
9
CR
T1R
T2R
CB
T1B
T2B
7
6
5
4
3
2
1
0
30 DAS
60 DAS
90 DAS
94
d) Protein
Table 20 shows the protein content in the leaves of Vigna radiata
(Figure 12) and Vigna mungo (Figure 13) grown in red and black soil using 25%
FIGURE 12
PROTEIN CONTENT IN THE LEAVES OF Vigna radiata
Protein content (mg/g)
6
5
CR
T1R
T2R
CB
T1B
T2B
4
3
2
1
0
30 DAS
60 DAS
90 DAS
FIGURE 13
PROTEIN CONTENT IN THE LEAVES OF Vigna mungo
8
CR
T1R
T2R
CB
T1B
T2B
6
5
4
3
2
1
0
30 DAS
60 DAS
90 DAS
95
2.22 0.44
1.82 0.76
T2B
2.94 0.38
4.06 0.34
4.91 0.63
3.24 0.64
4.85 0.25
5.26 0.38
90
2.72 0.22
3.48 0.75
3.65 0.68
3.70 0.25
4.85 0.89
4.96 0.67
30
0.11
3.90 0.77
4.32 0.62
4.62 0.35
5.20 0.64
5.64 0.37
5.72 0.65
60
Vigna mungo
5.90 0.97
5.21 0.68
5.30 0.28
5.17 0.47
5.86 0.67
6.82 0.37
90
96
96
0.12
3.88 0.35
4.25 0.53
3.15 0.34
CB
2.12 0.88
2.93 0.65
2.06 0.21
T2R
T1B
4.10 0.60
4.75 0.70
3.89 0.35
3.11 0.41
60
30
T1R
CR
Groups
CD (0.05)
Black
soil
Red soil
Soil
Vigna radiata
PROTEIN CONTENT (mg/g) IN THE LEAVES OF Vigna radiata AND Vigna mungo GROWN IN RED AND
BLACK SOIL USING DILUTED TANNERY EFFLUENT
TABLE 20
Protein content in the leaves recorded significant reduction (p<0.05)

compared to the control plants. Similar results were obtained by Malla and
Mohanty (2005), who stated that there was a negative correlation between the
effluent treatment and biochemical parameters such as chlorophyll, protein and
sugar. Vajpayee et al. (2001) observed a reduction in protein content and an
inhibition of nitrogenase activity in Vallisneria spiralis plant due to the presence
of chromium in the effluent.
Leaf part of R.sativus and B.nigra showed lower protein content in waste
water irrigated samples. Guo et al. (2007) reported that stress induced a decline
in soluble protein contents in plants but increase in soluble sugar contents.
There was a considerable reduction in the level of protein, lipid and
carbohydrate content in the leaves of plant treated with various concentrations
of effluent in a study conducted by Azmat et al. (2010). Gill and Sago (2010)
reported reduction of carbohydrate and protein content in turnip plants due to
higher concentration of cadmium, chromium and lead.
Reduction in protein content may be due to degradation by proteases.
High salinity was found to decrease protein content. The tannery effluent rich in
chromium might have caused the stress and thereby reduced the protein
content in the leaves.
4.2.2.2. Biochemical parameters in the seeds of Vigna radiata and Vigna
mungo
Total phenol, carotenoid, carbohydrate and protein contents were
analysed in the seeds of both the plants grown with the effluent.
1) Total phenol and carotenoid
Table 21 shows the total phenol (Figure 14) and carotenoid (Figure 15)
contents in the seeds of Vigna radiata and Vigna mungo grown in red and black
soil using 25% and 50% tannery effluent.
97
0.27
12.23 0.2
13.33 0.3
13.92 0.4
12.13 0.2
13.22 0.3
13.81 0.4
Vigna mungo
0.80 0.01
0.85 0.02
0.88 0.01
0.88 0.01
0.90 0.02
0.95 0.01
Vigna radiata
0.52
1.05 0.02
1.08 0.02
1.10 0.02
0.09 0.01
1.11 0.02
1.13 0.02
Vigna mungo
Carotenoid
98
98
11.83 0.1
13.01 0.3
CB
T2B
11.12 0.1
T2R
12.33 0.2
12.0 0.1
T1R
T1B
12.95 0.2
Vigna radiata
Total phenol
CR
Groups
CD ( 0.05)
Black soil
Red soil
Soil
TOTAL PHENOL AND CAROTENOID CONTENT (mg/g) IN THE SEEDS OF Vigna radiata AND Vigna mungo
GROWN IN RED AND BLACK SOIL USING DILUTED TANNERY EFFLUENT
TABLE 21
FIGURE 14
TOTAL PHENOL CONTENT IN THE SEEDS OF Vigna radiata
AND Vigna mungo
Total phenol content (mg/g)
18
16
CR
T1R
T2R
CB
T1B
T2B
14
12
10
8
6
4
2
0
Vigna radiata
Vigna mungo
FIGURE 15
CAROTENOID CONTENT THE IN SEEDS OF Vigna radiata
AND Vigna mungo
1.4
Carotenoid content (mg/g)
1.2
CR
T1R
T2R
CB
T1B
T2B
1
0.8
0.6
0.4
0.2
0
Vigna radiata
Vigna mungo
99
Total phenol content of the seeds of T1R plants of Vigna radiata and
Vigna mungo were 12 and 13.22 mg/g respectively. The T2R plants had
11.2 and 12.13 mg/g in Vigna radiata and Vigna mungo respectively. Thus there
was a significant decrease in total phenol content in seeds at (p<0.05) level.
Similar trend was observed in both the plants when grown with black soil.
A decrease in phenol content was observed in rice seedlings on
treatment with sewage sludge amendments in a study conducted by Singh and
Agrawal (2010).
The carotenoid content in the seeds of T1R of Vigna radiata and Vigna
mungo plants were found to be 0.90 and 1.11 mg/g respectively. T2R recorded
a value of 0.88 and 0.09 mg/g when grown in red soil whereas in black soil the
T1B and was found to be 0.85 and 1.08 mg/g and that of T2B was recorded
as 0.80 and 1.05 mg/g in Vigna radiata and Vigna mungo respectively. The
carotenoid contents in the seeds of Vigna radiata and Vigna mungo showed no
significant reduction when compared with control seeds.
According to the results of the study done by Okamoto et al. (2001), the
exposure to metals increase the activity of SOD and peroxidase but no
significant changes were detected in carotenoid contents of G. Polyedra which
was in agreement with our results.
b) Carbohydrate and protein
Table 22 shows the carbohydarate (Figure 16) and protein (Figure 17)
contents in the seeds of Vigna radiata and Vigna mungo grown in red and black
soil using 25% and 50% tannery effluent.
Carbohydrate and protein contents in the seeds of Vigna radiata and
Vigna mungo showed no significant reduction when grown in 25% and 50%
effluent.
100
14.34
301 8.86
302 8.73
305 10.66
310 9.48
315 10.32
321 11.2
Vigna mungo
235 5.82
237 5.68
241 4.26
237 6.43
240 5.05
244 6.45
Vigna radiata
10.59
224 5.0
230 4.69
233 6.23
230 5.39
231 6.13
234 5.74
Vigna mungo
Protein
101
101
319 9.63
T2B
328 11.29
CB
320 8.47
321 9.65
T2R
T1B
326 10.3
330 12.57
Vigna radiata
Carbohydrate
T1R
CR
Groups
CD ( 0.05)
Black soil
Red soil
Soil
CARBOHYDARATE AND PROTEIN CONTENT (mg/g) IN THE SEEDS OF Vigna radiata AND Vigna mungo
TABLE 22
FIGURE 16
CARBOHYDRATE CONTENT IN THE SEEDS OF Vigna radiata
AND Vigna mungo
350
340
CR
T1R
T2R
CB
T1B
T2B
330
320
310
300
290
Vigna radiata
Vigna mungo
FIGURE 17
PROTEIN CONTENT IN THE SEEDS OF Vigna radiata
AND Vigna mungo
255
CR
T1R
T2R
CB
T1B
T2B
250
245
240
235
230
225
220
215
210
205
200
Vigna radiata
Vigna mungo
102
The highest amount of protein content in Vigna mungo was recorded at

10% sago effluent concentration and the lowest content was recorded at 75%
(Sivaraman and Tamizhiniyan, 2005). The same trend was observed in paddy
seedling under tannery effluent treatment by Lakshmi and Sundaramurthy,
(2001).The increase in protein content at lower concentration of effluents could
be due to adsorption of most of the nitrogen by plants. The diluted effluent
reduces the salinity and enhances the plant growth.
c) Seed protein profile of Vigna radiata and Vigna mungo
Legume seeds have a high protein content compared to other
sources of plant proteins (Abou-El-Enain, 2002) and are highly stable and
unaffected by environmental conditions. Therefore electrphoretic technique for
analysis of total seed storage protein have been recognized as a valid method
(Emre et al., 2006; Tamilselvi, 2010).
Plates 5 and 6 show the polyacrylamide gel electrophoresis banding
pattern of the seed proteins of Vigna radiata and Vigna mungo respectively. The
banding patterns of seeds collected from control plants and the plants irrigated
with 25% and 50% effluent and grown in red soil and black soil are given in
lanes 2-7. The banding of protein marker of range 14.3 to 97.3 KDa is shown
in lane 1.
The storage protein profile of seeds of Vigna radiata (CR and CB)
showed 11 of bands with molecular weights 16.5 to 84.8 KDa, having 2 dense,
5 medium and 4 light bands. With respect to Vigna mungo (CR and CB) the total
bands were 12 with molecular weight ranging from 16.8 to 84.3 having 3 dense
5 medium and 4 light bands. The seeds of Vigna radiata experimental plants
namely T1R, T2R, T1B and T2B showed 10, 9, 8 and 7 bands respectively. The
molecular weight of T1R ranged from 16.5 to 84.8 KDa, while that of T2R
ranged from 19.3 to 69.1KDa. Similarly seeds of T1B ranged from 19.3 to 78.8
and T2B ranged from 29.2 to 69.1 KDa , which shows that the plants grown
with 50% effluent was found to have a stress on protein pattern.
103
PLATE 5
SEED PROTEIN PROFILE OF Vigna radiata
Lane 7
Lane 1
- Marker (M)
Lane 2
- Control red soil (CR)
Lane 3
- Red soil with 25% effluent (T1R)
Lane 4
Lane 5
- Control black soil (CB)
Lane 6
- Black soil with 25% effluent (T1B)
Lane 7
104
PLATE 6
SEED PROTEIN PROFILE OF Vigna mungo
Lane 7
Lane 1
- Marker (M)
Lane 2
- Control red soil (CR)
Lane 3
Lane 4
Lane 5
- Control black soil (CB)
Lane 6
Lane 7
105
The seeds of Vigna mungo experimental plants namely T1R, T2R, T1B
and T2B showed 12, 7, 10 and 7 bands respectively. The molecular weight of
T1R ranged from 16.8 to 84.3, while that of T2R ranged from 20.1 to 84.3 KDa .
Similarly seeds of T1B ranged from 18.3 to 84.3 KDa and T2B ranged from
29.8 to 84.3 KDa, which shows that the plants grown with 50% effluent was
found to have a stress on protein pattern. But the seeds of both the plants
grown with 25% effluent were not affected by the translocation of chemicals and
metals into the plants.
From the study it was found that the protein banding pattern was not
affected by growth with 25 % diluted tannery effluent however a differential
banding pattern was observed in the seeds of the plants grown with
50%
effluent.
According to Danimihardja and Lester (1974) it is genetically determined
that development of seeds always get priority over all physiological processes in
any plant. Hence, Vigna radiata and Vigna mungo plants were found to be
tolerant to contaminations, and the seed proteins were also found to be
unaltered in our study.
It could be derived from our study that the seeds of the plants grown with
25% tannery effluent did not show any modification and those grown with 50%
effluent showed a slight alteration in their molecular weights compared to the
control plants.
4.2.2.3 Enzymic and non enzymic antioxidants in the leaves and seeds of
Vigna radiata and Vigna mungo
Plants are rich sources of natural antioxidants that play a vital role in the
prevention or progression of the degenerative diseases. The consumption of
fruits, vegetables and herbs rich in antioxidants is associated with a decline in
the incidence of degenerative diseases and cancer (Harish et al., 2005).
106
Plants possess two very efficient antioxidant defense systems:

the enzymic which includes catalase, peroxidase, superoxide dismutase,
polyphenol oxidase, glutathione reductase and the non enzymic antioxidant
defense systems such as ascorbic acid, glutathione, tocopherol and
carotenoids. Both allow scavenging of reactive oxygen species leading to
protection of plant cells from oxidative damage (Blokhina et al., 2003;
Gratao et al., 2005). Indeed, activities of antioxidant enzymes have been
detected in various cellular organelles of various plant species. These
antioxidant enzymes were found in various compartments of the plant leaf cell,
eg: superoxide dismutase (SOD) found in chloroplasts (Mittova et al., 2000). It is
found that people who eat fruits and vegetables rich in polyphenols
and anthocyanins have a lower risk of cancer, heart disease and some
neurological diseases (Stanner et al., 2004). Antioxidants can cancel out the cell
damaging effects of free radicals. These compounds might prevent conditions
such as macular degeneration, suppressed immunity to poor nutrition and
neurodegeneration which is caused by oxidative stress (Wang et al., 2005).
The term antioxidant originally was used to refer to a chemical that
prevented the consumption of oxygen. Research into how vitamin E prevents
the process of lipid peroxidation led to the identification of antioxidants as
reducing agents that prevent oxidative reactions, often by scavenging reactive
oxygen species before they can damage cells (Wolf, 2005).
Heavy metals induce oxidative stress by generating free radicals and
toxic reactive oxygen species. These species react with lipids, proteins,
pigments and nucleic acids and cause lipid peroxidation, membrane damage
and inactivation of enzymes, thus affecting the cell viability. The deleterious
effects resulting from the cellular oxidative state may be alleviated by the
enzymatic and non enzymatic antioxidant machinery of the plant (Mittler 2002;
Sharma and Agrawal, 2005).
In the present study certain enzymic and non enzymic antioxidants were
analysed in the leaves and seeds of both the plants grown with the effluent.
107
Enzymic antioxidants
The enzymic antioxidants and free radical scavengers may provide a
defensive mechanism against the deleterious actions of reactive oxygen species
(ROS). Some of the antioxidant enzymes that are found to provide protection
against the ROS are superoxide dismutase, catalase, peroxidase, glutathione
reductase and ascorbate oxidase (Rani et al.,2004). Free radicals are implicated
in several degenerative diseases such as arteriosclerosis, diabetes, arthritis,
cancer and aging. The harmful effects of free radicals on living systems could
be attenuated by antioxidants that scavenge the free radicals. Uptake of any
excess heavy metal induces a deficiency of essential nutrients affecting the
cationic balance at subcellular level altering certain enzymes (Sharma and
Chettri, 2008).
The deleterious effects from the cellular oxidative state may be alleviated
by the enzymic and non enzymic antioxidant machinery of the plant. The
antioxidants of legume leaves and seeds have been examined in considerable
detail (Matamoras et al., 2003; Palma et al., 2006). The enzymic antioxidants
analysed in the seeds and leaves of Vigna radiata and Vigna mungo plants
grown in red and black soils using 25% and 50% tannery effluent were catalase,
peroxidase, super oxide dismutase and glutathione reductase.
a.) Catalase and Peroxidase
Catalases are enzymes that catalyze the conversion of hydrogen
peroxide to water and oxygen using either iron or manganese as cofactor
(Chelikani et al., 2004) and an enzymic antioxidant which protects the tissue
from highly reactive hydroxyl radical (Himer et al., 2002; Dash et al., 2007)
Peroxidases are widely distributed in plant tissues and are of immense
physiological interest because of their association with numerous catalytic
functions. Numerous functions have been proposed and the most important
among these are the ability to oxidize indole-3-acetic acid, ethylene
biosynthesis, hydroxylation of proline, lignifications and disease resistance
(Hiraga et al., 2001).
108
2.16 0.09
2.01 0.10
1.81 0.21
CB
T1B
T2B
1.04 0.08
1.94 0.11
2.01 0.07
0.82 0.03
1.78 0.05
0.98 0.23
0.21 0.03
1.32 0.06
0.09 0.02
1.08 0.6
Vigna
mungo
1.18 0.08
0.29
1.02 0.5
1.32 0.14
1.48 0.01
0.07 0.01
1.22 0.1
Vigna
radiata
1.32 0.08
Seeds
0.130.05
0.120.02
0.110.02
0.080.02
0.070.01
0.170.01
0.160.03
0.120.04
0.170.05
0.160.04
0.04
0.180.01
0.170.01
0.130.02
0.170.03
0.150.05
109
109
0.06
0.310.02
0.280.04
0.220.03
0.190.01
0.210.02
Seeds
Vigna
Vigna
radiata
mungo
0.130.02 0.110.03
Peroxidase (Units /g)

Leaves
Vigna
Vigna
radiata
mungo
0.150.03 0.060.01
Catalase units - amount of enzyme required to decrease the optical density by 0.05 units.
Peroxidase units change in absorbance at 450 nm/ min.
0.21
1.78 0.04
T2R
CD( 0.05)
1.92 0.03
Leaves
Vigna
Vigna
radiata
mungo
2.01 0.05 1.98 0.08
T1R
CR
Groups
Catalase (Units/g)
CATALASE AND PEROXIDASE ACTIVITIES IN THE LEAVES AND SEEDS OF Vigna radiata AND Vigna mungo
TABLE 23
Table 23 shows the catalase (Figure 18) and peroxidase (Figure 19)
activities in the leaves and seeds of Vigna radiata and Vigna mungo grown in
red and black soil using 25% and 50% tannery effluent.
FIGURE 18
Units / g
CATALASE ACTIVITIES IN THE LEAVES AND SEEDS OF

Vigna radiata AND Vigna mungo
2.5
CR
T1R
T2R
CB
T1B
T2B
1.5
0.5
0
Vigna radiata
Vigna mungo
Vigna radiata
Leaves
Vigna mungo
Seeds
FIGURE 19
PEROXIDASE ACTIVITIES IN THE LEAVES AND SEEDS OF
0.4
CR
T1R
T2R
CB
T1B
T2B
0.35
Units / g
0.3
0.25
0.2
0.15
0.1
0.05
0
Vigna radiata
Vigna mungo
Leaves
Vigna radiata
Vigna mungo
Seeds
110
The catalase activity was significantly reduced in the leaves of Vigna

radiata and Vigna mungo when grown with 50% effluent. Seeds also recorded
significant reduction in the catalase activity when both the plants were grown
with 50% effluent.
Peroxidase activity of the leaves and seeds of Vigna radiata showed
significant increase on growth with both the types of effluents. Vigna mungo
plants were found to exhibit increased activities of peroxidase in both leaves
and seeds compared to the control plants.
Singh and Agrawal (2007) observed an increase in the activity of
peroxidase in root, stem and leaf of Beta vulgaris plants grown in sewage
sludge amended pots. Toxic doses of zinc, uranium and cadmium inhibited
shoot growth but increased the glucose 6 phosohate dehydrogenase activity
and peroxidase activity in the leaves of dwarf beans (Vandenhove et al., 2006).
Many authors have shown a concomitant increase in the peroxidase
activity in plants after prolonged exposure to high cadmium concentration
(Tamas et al., 2003; Chaoui et al., 2004; Saffar et al., 2009).
b) Superoxide dismutase and Glutathione reductase
Superoxide dismutase (SOD) enzymes are present in all aerobic cells
and in extra cellular fluids (Johnson and Giulivi, 2005). This contains metal ion
cofactors like zinc, manganese or iron. In plants SOD isozymes are present in
the cytosol and mitochondria, with an iron SOD found in chloroplast which is not
present in vertebrates and yeast. SOD catalyzes the dismutation of superoxide
into oxygen and hydrogen peroxide (Alscher et al., 2003).
Glutathione reductase is ubiquitous NADPH dependant enzyme which
catalyses the reduction of oxidized glutathione present in cells. It has been well
documented that the activity of glutathione reductase altered due to metal stress
(Schutzendubel and Polle, 2002; Meloni et al., 2003).
111
5.41 0.23
6.11 0.24
7.22 0.56
CB
T1B
T2B
7.5 0.23
6.2 0.20
5.73 0.20
8.21 0.18
7.01 0.19
1.12
7.91 0.09
6.5 0.17
5.8 0.13
9.71 0.11
8.5 0.18
3.2 0.28
2.1 0.23
1.13 0.21
0.92 0.26
2.9 0.22
2.21 0.36
1.4
2.82 0.38
1.0 0.35
3.0 0.26
2.3 0.28
2.5 1.16
1.9 1.24
1.4 1.52
2.4 1.61
2.0 0.51
0.98
2.8 1.10
2.0 0.23
1.2 0.34
2.9 0.36
2.2 0.21
Glutathione reductase (Units /g)

Leaves
Seeds
Vigna
Vigna
Vigna
Vigna
radiata
mungo
radiata
mungo
1.8 0.29
1.0 0.27
1.3 0.55 1.5 0.30
112
112
Glutathione reductase- Unit- moles of glutathione consumed /min/g of the sample
SOD- Unit- the amount that causes 50% reduction in the extent of NBT oxidation.
7.2 0.10
6.8 0.11
5.22 0.13
8.71 0.16
7.62 0.14
1.08
9.02 0.82
T2R
CD( 0.05)
8.8 0.08
T1R
CR
Groups
Superoxide dismutase (Units/g)

Leaves
Seeds
Vigna
Vigna
Vigna
Vigna
radiata
mungo
radiata
mungo
7.2 0.12
6.01 0.15 7.8 0.19 6.51 0.21
SUPEROXIDE DISMUTASE AND GLUTATHIONE REDUCTASE ACTIVITIES IN THE LEAVES AND SEEDS OF
Vigna radiata AND Vigna mungo GROWN IN RED AND BLACK SOIL USING
TABLE 24
Table 24 shows the superoxide dismutase (Figure 20) and glutathione

reductase (Figure 21) activities in the leaves and seeds of Vigna radiata and
Vigna mungo grown in red and black soil using 25% and 50% tannery effluent.
FIGURE 20
SUPEROXIDE DISMUTASE ACTIVITIES IN THE LEAVES AND
SEEDS OF Vigna radiata AND Vigna mungo
12
CR
T1R
T2R
10
CB
T1B
T2B
Units / g
8
6
4
2
0
Vigna radiata
Vigna mungo
Vigna radiata
Leaves
Vigna mungo
Seeds
FIGURE 21
GLUTATHIONE REDUCTASE ACTIVITIES IN THE LEAVES AND
SEEDS OF Vigna radiata AND Vigna mungo
4
3.5
CR
T1R
T2R
CB
T1B
T2B
Units / g
3
2.5
2
1.5
1
0.5
0
Vigna radiata
Vigna mungo
Leaves
Vigna radiata
Vigna mungo
Seeds
113
There was significant increase in the SOD activities in the leaves of

Vigna radiata and Vigna mungo plants on treatment with effluents. Seeds of
both the plants showed a significant increase in SOD activities on treatment with
50% effluent.
The high SOD activity indicates that this plant can efficiently detoxify toxic
superoxide radicals produced by the accumulated chromium and it has been
associated with stress tolerance in plants because it neutralizes the reactivity of
oxygen. It has been well documented that SOD activity has a protective role in
heavy metal accumulated plants (Gratao et al., 2005 ; Labra et al., 2006).
Glutathione reductase activity in the leaves of Vigna radiata and Vigna
mungo showed a significant increase when the concentration of the effluent was
increased correspondingly. Seeds of plants grown with 50% effluent showed
significant increase of glutathione reductase activity compared to that of control
plants. Increase in glutathione reductase activity might be due to the result of
reaction of metal with sulfhydral groups.
A similar study conducted by Diwan et al. (2010a) reported that SOD
activity of Vigna radiata was increased 7- folds on chromium exposure and
glutathione reductase increased significantly over untreated plants.
Non enzymic antioxidants
The non enzymic antioxidants analysed in the leaves and seeds of Vigna
radiata and Vigna mungo grown in red and black soils using 25% and 50%
tannery effluent were ascorbic acid, riboflavin, tocopherol and flavonoids.
a) Ascorbic acid and riboflavin
Ascorbic acid has antioxidant activity and it reduces oxidizing
substances such as hydrogen peroxide (Duarte and Lunec, 2005). It can also
reduce metal ions which lead to the generation of free radicals through the
Fenton reaction (Valko et al., 2005).
114
9.02 0.13
5.41 0.30
6.11 0.51
7.22 0.74
T2R
CB
T1B
T2B
7.5 0.3
6.2 0.6
5.73 0.4
8.21 0.4
7.01 0.3
6.51 0.2
2.24
7.91 0.4
6.5 0.7
5.8 0.6
9.71 0.3
8.5 0.2
7.8 0.1
Seeds
Vigna
Vigna
radiata
mungo
3.2 0.03
2.82 0.02
1.0 0.03
3.0 0.07
2.3 0.06
1.8 0.05
0.4
2.1 0.06
1.13 0.07
0.92 0.02
2.9 0.04
2.21 0.05
1.0 0.09
2.5 1.09
1.9 1.08
1.4 1.04
2.4 1.05
2.0 1.05
1.5 1.03
0.32
2.8 1.04
2.0 0.05
1.2 0.02
2.9 0.05
2.2 0.06
1.3 0.03
Riboflavin contents
Leaves
Seeds
Vigna
Vigna
Vigna
Vigna
radiata
mungo
radiata
mungo
115
115
7.2 0.8
6.8 0.7
5.22 0.6
8.71 0.4
7.62 0.2
6.01 0.3
2.1
8.8 0.23
T1R
CD( 0.05)
7.2 0.21
Leaves
Vigna
Vigna
radiata
mungo
CR
Groups
Ascorbic acid
ASCORBIC ACID AND RIBOFLAVIN CONTENT (mg/g) IN THE LEAVES AND SEEDS OF Vigna radiata
AND Vigna mungo GROWN IN RED AND BLACK SOIL USING DILUTED
TANNERY EFFLUENT
TABLE 25
In cells ascorbic acid is maintained in its reduced form by reaction with

glutathione, which can be catalyzed by protein disulphide isomerase and
glutaredoxins. Ascorbic acid is a reducing agent that can reduce and thereby
neutralize oxygen species such as hydrogen peroxide (Padayatty et al., 2003).
In addition to its direct antioxidant effects, ascorbic acid is also a substrate for
the antioxidant enzyme ascorbate peroxidase, a function that is particularly
important in stress resistance in plants (Shigeoka et al., 2002).
Table 25 shows the ascorbic acid (Figure 22) and riboflavin (Figure 23)
contents in the leaves and seeds of Vigna radiata and Vigna mungo grown in
red and black soil using 25 % and 50 % tannery effluent.
Ascorbic acid content in the leaves of both the plants reported a
significant increase when grown with the effluent whereas in seeds,
the significant increase was noted only with 50% effluent. According to
Guo et al. (2005) ascorbic acid was found to increase in all waste water
irrigated plants than control.
FIGURE 22
Ascorbic acid (mg/g)
ASCORBIC ACID CONTENT IN THE LEAVES AND SEEDS OF

12
CR
T1R
T2R
10
CB
T1B
T2B
8
6
4
2
0
Vigna radiata
Vigna mungo
Leaves
Vigna radiata
Vigna mungo
Seeds
116
FIGURE 23
RIBOFLAVIN CONTENT IN THE LEAVES AND SEEDS OF
3.5
CR
T1R
T2R
CB
T1B
T2B
Riboflavin (mg/g)
3
2.5
2
1.5
1
0.5
0
Vigna radiata
Vigna mungo
Leaves
Vigna radiata
Vigna mungo
Seeds
Gupta et al. (2010) reported that heavy metal accumulation showed a

varied response in plant species and also increases the ascorbic acid contents.
In a study conducted by Singh and Agrawal (2009), application of sewage
sludge increased the ascorbic acid contents in Abelmochus esculentus plants.
The riboflavin content in the leaves and seeds Vigna radiata showed
a significant increase in plants grown with 25 and 50% effluent. Similar
observation was noticed in the leaves and seeds of Vigna mungo plants.
b) Flavonoid and tocopherol
Table 26 shows the flavonoid (Figure 24) and tocopherol (Figure 25)
contents in the leaves and seeds of Vigna radiata and Vigna mungo grown in
red and black soil using 25 % and 50 % tannery effluent.
117
192 2.8
209 3.1
170 2.9
199 2.7
213 3.0
T1R
T2R
CB
T1B
T2B
4.8
183 1.6
156 1.7
135 1.6
172 1.4
149 1.8
120 1.9
175 2.32
138 2.17
114 2.32
166 2.16
132 2.15
105 2.9
4.98
199 3.6
176 3.1
155 2.8
194 2.7
162 2.8
148 2.6
Seeds
Vigna
Vigna
radiata
mungo
2.4 0.04
1.7 0.08
2.12 0.06
2.03 0.04
1.84 0.05
2.0 0.06
1.91 0.09
1.7
2.11 0.03
1.98 0.03
2.2 0.04
1.93 0.02
1.9 0.08
Leaves
Vigna
Vigna
radiata
mungo
1.1
2.32 0.08 2.04 0.02
1.02 0.09 1.92 0.03
0.84 0.07 0.72 0.09
2.14 0.06 1.94 0.06
1.98 0.05 0.85 0.05
0.73 0.04 0.63 0.08
Vigna
mungo
Seeds
Vigna
radiata
Tocopherol
118
118
CD( 0.05)
162 2.1
Leaves
Vigna
Vigna
radiata
mungo
CR
Groups
Flavonoid
FLAVONOID (g/g) AND TOCOPHEROL (mg/g) CONTENT IN THE LEAVES AND SEEDS OF Vigna radiata AND
Vigna mungo GROWN IN RED AND BLACK SOIL USING DILUTED TANNERY EFFLUENT
TABLE 26
FIGURE 24
FLAVONOID CONTENT IN THE LEAVES AND SEEDS OF
250
CR
T1R
T2R
CB
T1B
T2B
Flavonoid ( g/g)
200
150
100
50
0
Vigna radiata
Vigna mungo
Vigna radiata
Leaves
Vigna mungo
Seeds
FIGURE 25
TOCOPHEROL CONTENT IN THE LEAVES AND SEEDS OF
3
CR
T1R
T2R
CB
T1B
T2B
Tocopherol (mg/g)
2.5
2
1.5
1
0.5
0
Vigna radiata
Vigna mungo
Leaves
Vigna radiata
Vigna mungo
Seeds
119
Dietary flavonoid makes an important contribution to health and

cardiovascular system (Lazarus et al., 2000). Flavonoids are secondary
metabolites and functions as chelators for metals (Korkina, 2007). It has a vast
array of functions in plants including antioxidant activity (Havsteen, 2002).
Tocopherol is the most important lipid soluble antioxidant and it protects
membranes from oxidation by reacting with lipid radicals produced in the lipid
peroxidation chain reaction (Traber and Atkinson, 2007). This removes the free
radicals intermediates and prevents the propagation reaction. The oxidized
tocopherol radicals produced in this process may be recycled back to the
active reduced form through reduction by ascorbate, retinol and ubiquinol
(Wang and Quinn, 1999).
Flavonoid content in the leaves and seeds of both the plants had a
significant increase (p<0.05) when grown with both effluent concentrations than
that of control plants. Keilig and Muller (2009) observed an increase in flavonoid
content in Arabidopsis thaliana on growth with different concentrations of
cadmium and zinc.
The tocopherol content of the T1R, T2R, T1B and T2B plants showed no
significant difference on comparison with control plants. Tocopherol content of
the seeds of T2R and T2B plants recorded a significant increase.
According to Gajewska and Sklodowska (2007), the tocopherol content
increased in shoots of wheat seedlings on treatment with nickel.
Menach et al. (2004), reported that leguminous plants are rich sources of
flavonoids. Flavonoids are postulated to play a pivotal role in adaptation of
producer legumes to their biological environments both as defensive
compounds and chemical signals in symbiotic nitrogen fixation with rhizobia
(Aoki et al., 2000)
120
4.2.2.4. Metal contents in various parts of Vigna radiata and Vigna mungo
plants
The use of industrial effluents carrying a high load of heavy metals such
as zinc, chromium and nickel for irrigation of crops produces adverse effect on
plant growth (Pandey et al., 2008). Essential heavy metals like iron, manganese,
zinc and copper in all higher plants were found to be absorbed and accumulated
in plant tissues based on their solubility, concentration and availability of
different ions in the soil (Sharma and Chettri, 2008).
Accumulation and exclusion are two basic strategies by which plants
respond to elevated concentration of heavy metals. It is known that some plants
can survive quite well under elevated metal conditions without the hyper
accumulation characteristic. These plants appear to tolerate metals in the
environment by using a variety of mechanism including exudation of compounds
that complex metals in the contaminated environment, thereby preventing their
entry into the root (Vogel-Mikus et al., 2005).
Chromium
Chromium is known to be highly toxic to biological systems. The
morphological growth parameters like germination percentage, root length,
shoot length, fresh weight and dry weight of black gram seedling were
decreased when chromium concentration was increased (Chidambaram et al.,
2009).
Tables 27 and 28 show accumulation of chromium in roots, shoots,
leaves and seeds of the plants Vigna radiata (Figure 26) and Vigna mungo
(Figure 27) grown in red soil and black soil using 25% and 50% tannery effluent.
In 25 % effluent, the chromium content in the roots of both the plants
were found to be 4.8 mg/g and 4.6 mg/g respectively. The chromium content
was decreased to 3.84 mg/g and 2.1 mg/g in shoots which indicates the upward
translocation of chromium to shoots.
121
0.063
CD (0.05)
T2
122
0.046
122
0.2 0.01 3.97 0.63 5.35 0.98 0.2 0.01
Seeds : Not detectable
T1
SHOOTS
0.2 0.01 3.84 0.52 4.93 0.75 0.1 0.01
C- Control soil , T1- Soil with 25% effluent, T2 - Soil with 50% effluent.
0.4 0.01 4.9 0.92 5.9 0.97
T2
Black
soil
T1
0.3 0.01 4.8 0.89 5.4 1.04
ROOTS
Red soil
SOIL
T2
0.53
2.86 0.41 3.92 0.47
2.53 0.35 3.21 0.39
T1
LEAVES
CHROMIUM CONTENT (mg/g) IN VARIOUS PARTS OF Vigna radiata GROWN IN RED SOIL AND
BLACK SOIL USING DILUTED EFFLUENT
TABLE 27
T1
0.4 0.03 2.1 0.03
SHOOTS
123
0.046
6.32 0.7 0.5 0.02 2.54 0.3
5.3 0.7
T2
0.063
0.9 0.03 4.5 0.6
Black
soil
CD (0.05)
0.6 0.03 4.6 0.8
T1
Red soil
ROOTS
123
4.1 0.4
2.9 0.4
T2
T1
T2
0.2 0.03
0.53
2.2 0.5
3.12 0.4
0.2 0.03 1.43 0.02 1.53 0.2
LEAVES
CHROMIUM CONTENT (mg/g) IN VARIOUS PARTS OF Vigna mungo GROWN IN RED SOIL
AND BLACK SOIL USING DILUTED EFFLUENT
TABLE 28
FIGURE 26
CHROMIUM CONTENT IN VARIOUS PARTS OF Vigna radiata
7
Root
Shoot
Leaf
mg / g
5
4
3
2
1
0
C
T1
T2
Red soil
T1
T2
Black soil
FIGURE 27
CHROMIUM CONTENT IN VARIOUS PARTS OF Vigna mungo
8
Root
Shoot
Leaf
7
6
mg / g
5
4
3
2
1
0
C
T1
Red soil
T2
T1
T2
Black soil
124
Leaves of both plants recorded a value of 2.53 mg/g and 1.43 mg/g
respectively whereas chromium was not detected in grains. The same trend was
noted when the plants were grown with 50% effluent except that seed contained
negligible amounts of chromium. Calheiros et al. (2008a) reported the effect of
tannery waste water in the development of P.australis as the accumulation
of the metal in the plant was found to be in the following decreasing order
root > shoot> leaf. The concentration of chromium in the leaves, shoots and
roots increased with the concentration applied. According to Weis and Weis
(2004) the degree of upward translocation depends upon the plant species,
metal and several environmental conditions.
Several workers have documented high levels of chromium accumulation
in roots than in the top portions of the plant namely pod, leaves and stem which
was in agreement with our result (Ghosh and Singh, 2005 ; Yang et al., 2003).
Nickel
It is the essential heavy metal for plant growth and development. Under
normal conditions plants take up small quantities of nickel from soils. It can be
toxic to plants when its concentration in the soil is high. In plants under nickel
stress, the absorption of nutrients, root development and metabolism are
strongly retarded. Along with the toxicity symptoms in plants that develop later,
plant tissues are known to inhibit photosynthesis and transpiration (Zarkovic and
Blagojevic, 2009).
Tables 29 and 30 show the accumulation of nickel in roots, shoots,
leaves and seeds of the plants Vigna radiata and Vigna mungo grown in red soil
and black soil using 25% and 50% tannery effluent.
In Vigna radiata plants grown using 25 and 50% effluent, the nickel
content was found to be higher in roots compared to other parts of the plant The
value in grains was negligible. Similar trend was followed in Vigna mungo plants
also.
125
T1
T2
T1
SHOOTS
T2
T1
LEAVES
0.85
126
0.76
126
0.9 0.03 3.3 0.61 3.9 0.72 0.07 0.03 2.2 0.34 3.1 0.39
0.37
0.3 0.02 1.65 0.24
0.6 0.02 3.01 0.45 3.7 0.56 0.32 0.01 2.1 0.44 2.9 0.53 0.14 0.04 1.17 0.25
ROOTS
CD (0.05)
Black
soil
Red soil
Soil
NICKEL CONTENT (mg/g) IN VARIOUS PARTS OF Vigna radiata GROWN IN RED SOIL AND
TABLE 29
2.68 0.29
2.58 0.41
T2
T1
T2
T1
T2
0.3 0.02 2.9 0.18 2.7 0.21 0.1 0.03
SHOOTS
1.54 0.2
T1
LEAVES
0.34
127
0.27
127
0.20
0.8 0.04 2.5 0.21 3.0 0.23 0.6 0.02 1.9 0.15 2.4 0.22 0.5 0.11 1.48 0.30
0.5 0.03 3.2 0.24 3.5 0.27
ROOTS
CD (0.05)
Black
soil
Red soil
Soil
NICKEL CONTENT (mg/g) IN VARIOUS PARTS OF Vigna mungo GROWN IN RED SOIL AND
TABLE 30
1.95 0.4
1.68 0.3
T2
A similar observation was reported by Zarkovic and Blagojevic, (2009) in

their study on uptake if nickel by maize plants, where the concentration of nickel
was 8 times higher in roots than in shoots
Metals in tannery waste water occur in a complex form and vary in their
availability to the plant parts (Gupta and Sinha, 2007).
Zinc
Zinc is an essential micronutrient for all organisms and form active site for
various metalloenzymes. Excessive intake of zinc may lead to vomiting,
dehydration and other adverse effects. Industrial waste water rich in zinc
cadmium, chromium and other heavy metals pose a major threat to the
agricultural fields.
The plants irrigated with diluted form of industrial effluent do not face
much hazards as with the raw effluent which contains the heavy metals at a
higher concentration.
Tables 31 and 32 show the accumulation of zinc in roots, shoots, leaves
and seeds of the plants Vigna radiata and Vigna mungo grown in red soil and
black soil using 25% and 50% tannery effluent
Zinc contents of the plant Vigna radiata and Vigna mungo in various parts
were found to be higher than that of the control plants. Zinc content was higher
in roots but negligible in grain. The accumulation of zinc followed a decreasing
order from roots, shoots, leaves and grain.
Khilji and Barbeen (2008) observed the percentage reduction in the
amount of metals in different concentrations of tannery sludge after growing
H. Umbellata for 90 days. Akinola and Ekiyoyo (2006) studied that accumulation
of cadmium, lead and chromium in crop plants were high in roots compared to
other parts.
128
T1
T2
T1
SHOOTS
T2
T1
LEAVES
0.91
129
0.58
129
0.8 0.02 4.5 0.71 4.9 0.82 0.6 0.03 3.6 0.29 3.9 0.32 1.54 0.12
0.20
1.3 0.13
0.7 0.02 4.2 0.45 5.1 0.63 0.6 0.02 3.4 0.25 2.25 0.02 0.48 0.03 1.37 0.14
ROOTS
CD (0.05)
Black
soil
Red soil
Soil
ZINC CONTENT (mg/g) IN VARIOUS PARTS OF Vigna radiata GROWN IN RED SOIL AND
TABLE 31
2.9 0.19
1.0 0.21
T2
T1
T2
T1
0.89
CD (0.05)
130
0.61
130
0.18
0.9 0.04 4.7 0.62 5.1 1.81 0.7 0.03 3.8 0.29 4.1 0.22 0.57 0.12 2.10 0.19
T2
LEAVES
Black
soil
T1
SHOOTS
0.8 0.03 4.9 0.53 5.6 1.73 0.7 0.04 3.9 0.32 4.8 0.41 0.56 0.04 1.33 0.15
ROOTS
Red soil
Soil
ZINC CONTENT (mg/g) IN VARIOUS PARTS OF Vigna mungo GROWN IN RED SOIL AND
TABLE 32
2.18 0.20
1.72 0.16
T2
Cadmium
Cadmium is one of the most toxic heavy metals and is recognized for its
negative effect on the environment where it accumulates throughout the food
chain posing a serious threat to human health (Xiaomei et al., 2004).The uptake
of cadmium by roots and transport to the upper parts takes place in the upward
translocation mechanism (Huttova et al., 2006).
Cadmium has been recognised to have a negative impact on the
environment in high concentration. The presence of excessive amount of
cadmium in soil causes reduction in root growth, disturbance in mineral nutrients
and carbohytrate metabolism. They were found to reduce biomass production
due to the direct consequence of the inhibition of chlorophyll synthesis and
photosysnthesis. The stress caused by cadmium and zinc in the tannery effluent
could be reduced by diluting the effluent before application on the vegetative
crops.
Table 33 and 34 show the accumulation of cadmium in roots,
shoots, leaves and seeds of the plants Vigna radiata and Vigna mungo grown in
red soil and black soil using 25% and 50% tannery effluent.
Cadmium content in various plant parts of Vigna radiata in 50% effluent
in the present study was found to have the following order of accumulation as
3.1 mg/g, 2.5 mg/g and 2.25 mg/g in root, shoot and leaves respectively. But it
was not detectable in grains. The same trend was noticed in Vigna mungo
plants.
According to Sharma and Chettri (2008) cadmium and lead were found
to have accumulated in plant tissues. Although cadmium adversely affects plant
growth, root growth is severely affected and results in faster reduction of root
biomass compared to the shoot resulting in an increased shoot root biomass
ratio (Chandra et al., 2010).
131
T1
ROOTS
T2
T1
SHOOTS
T2
T1
LEAVES
C- Control soil, T1- Soil with 25% effluent, T2 - Soil with 50% effluent.
132
132
Black
1.09 0.14 2.9 0.16 3.9 0.27 0.7 0.05 2.8 0.19 3.1 0.29
soil
CD (0.05)
0.25
0.31
0.310.12
0.26
1.8 0.15
Red soil 1.05 0.11 2.8 0.19 3.1 0.21 0.82 0.07 2.27 0.16 2.5 0.24 0.24 0.05 1.9 0.14
Soil
CADMIUM CONTENT (mg/g) IN VARIOUS PARTS OF Vigna radiata GROWN IN RED SOIL
TABLE 33
2.90 0.23
2.25 0.25
T2
T1
ROOTS
T2
T1
SHOOTS
T2
T1
LEAVES
0.44
1.0 0.03 3.0 0.24

3.6 0.35
133
0.23
133
0.45
0.8 0.10 2.85 0.12 3.0 0.20 0.64 0.09 2.13 0.29
C- Control soil, T1- Soil with 25% effluent, T2- Soil with 50% effluent.
CD (0.05)
Black
soil
Red soil 1.04 0.07 2.35 0.12 3.8 0.32 0.71 0.11 2.2 0.09 2.7 0.18 0.03 0.96 1.31 0.24
Soil
CADMIUM CONTENT (mg/g) IN VARIOUS PARTS OF Vigna mungo GROWN IN RED SOIL AND
TABLE 34
2.62 0.36
2.12 0.41
T2
Stolt et al. (2006) opined that the rate of absorption and translocation of
cadmium varies from plant to plant and genetic variation exists in the
accumulation rate of cadmium in different parts of the plant.
Low grain cadmium accumulation was observed in many studies. The
mechanism of cadmium uptake, translocation and grain accumulation depends
on lower cadmium pools in leaves. Much of the cadmium was found to be
retained in the root cell walls during the growth with 25% effluent
The gradient of accumulation of heavy metals was found to be highest in
roots followed by stem, branches, leaves and then in grains or fruits as reported
by Sharma and Agrawal (2005). Fritioff and Gregor (2006) in their study on
distribution of heavy metals such as zinc, copper, lead and cadmium by leaves,
stems and roots of Potamogeton natans, found the highest accumulation in
roots than other parts in conformity with the present study. Angelova and Ivanov
(2008), in the work on distribution of heavy metals in Brassica Nigera, reported
that the metal content of the seeds were lower in comparision to other parts. It
follows the order roots> stems> leaves>fruit shells> seeds. Fruit shells act as a
barrier on their way towards the seeds.
In accordance with these observations the concentration of metals in our
study were found to be higher in roots followed by shoots and leaves, with least
concentrations in grain. The seed coat of the grains might have acted as a
barrier in Vigna radiata and Vigna mungo in condtions of soil contamination.
4.2.3. Histochemical observations of root sections of the selected plants
Plate 7 show the cross sections of root of Vigna radiata and Plate 8 show
that of Vigna mungo plants grown in 25% and 50% tannery effluent.
134
PLATE 7
HISTOCHEMICAL OBSERVATION OF ROOTS OF Vigna radiata
CR
CB
T1R
T1B
T2R
T2B
CR- Control with red soil, T1R- Red soil with 25% effluent, T2R- Red soil with 50% effluent, CB- Control
black soil, T1B- Black soil with 25% effluent, T2B- Black soil with 50% effluent
135
PLATE 8
HISTOCHEMICAL OBSERVATION OF ROOTS OF Vigna mungo
C1R
C1B
T1R
T1B
T2R
T2B
CR- Control with red soil, T1R- Red soil with 25% effluent, T2R- Red soil with 50% effluent, CB- Control
black soil, T1B- Black soil with 25% effluent, T2B- Black soil with 50% effluent
136
Localization of metals in various plant tissues and the concerned

anatomical variations in effluent cultivated plants was done by histochemical
staining method.
Both the plants grown with 25% effluent (T1R and T1B) did not show any
accumulation in their root tissues. Roots of T2R and T2B Vigna radiata plants
exhibited accumulation of heavy metal. Similarly the Vigna mungo plants grown
with 50% effluent (T2R and T2B) were found to have accumulations of metals in
the cross sectional study of roots.
It was observed that there were many sites of accumulation in the roots
of Vigna mungo plant compared to the roots of Vigna radiata plants indicating
that metals are fast absorbed in Vigna mungo plants than Vigna radiata plants.
An important reason for enhanced accumulation of chromium in the root may be
due to presence of organic acids in the root exudates which form complexes
with chromium, thereby making them available for the uptake by the root.
Histological changes in the root showed highly distorted piliferous layer
and cortex. Thickened cell walls of vessels and pith were noted. The distortion
of cells of various tissues may be the result of interferences with the cell division
or with cell elongation.
Though metal concentrations were found in leaves and shoots of both the
plants, the histochemical sections did not show any accumulation. As Shankar
et al. (2005) suggested, translocation of chromium from root to shoot is slow.
Pulford et al. (2001) in a study with temperate trees confirmed that chromium is
poorly taken up into the aerial parts and is predominantly held in the roots.
In our study, in both the species of Vigna chromium accumulation was
high in the roots compared to the stems, leaves and grains which was in
concomitment with the results obtained by Chandra et al. (2010).
137
4.2.4. Yield parameters

a. Number of nodules
In nodulating plants, nitrogen fixation is the primary route of nitrogen
nutrition and hence what happens to the nodules at the stimulated growth
conditions will have a profound effect on the overall growth of the plants
themselves. Leguminous plants exhibit differential response in nodulation to
heavy metal toxicity (Veliappan et al., 2002).
Table 35 and 36 show the number of nodules formed in plants Vigna
effluent concentrations respectively.
TABLE 35
NUMBER OF NODULES OF Vigna radiata GROWN IN RED SOIL
C1
Vigna radiata
30
60
90
10.0 4.08
19.0 0.82
21.0 0.80
T1
9.0 0.82
18.0 0.81
20.0 0.82
T2
8.0 1.63
17.0 0.82
19.0 0.80
C2
11.0 0.82
18.0 1.63
19.0 1.60
T3
10.0 0.82
16.0 6.24
16.0 1.63
T4
CD ( 0.05)
9.0 0.82
13.3 2.45
4.2
16.0 0.82
Soil
Red soil
Black soil
Groups

CR- Control with red soil, T1R- Red soil with 25% effluent, T2R- Red soil with 50% effluent,
CB- Control black soil, T1B- Black soil with 25% effluent, T2B- Black soil with 50% effluent
Number of nodules were increased with increase in the time point and
reached a maximum level on 60th day and sustained till 90th day in both the
plants. According to Geetha et al. (2008), the nodule formation in soybean
reached its peak during flowering stage and senescence of nodules occurred as
the plant matured.
138
TABLE 36
NUMBER OF NODULES OF Vigna mungo GROWN IN RED SOIL
Soil
Vigna radiata
30
60
90
11.0 1.63
15.0 4.08
16.0 1.63
9.0 0.79
13.0 2.45
14.0 3.27
8.0 0.82
12.0 1.63
13.0 2.45
12.0 1.61
16.0 1.63
16.0 0.82
11.0 4.08
15.0 3.27
14.0 3.25
10.0 0.80
14.0 4.08
13.0 2.45
5.1
Groups
C1
Red soil
T1
T2
C2
T3
Black soil
T4
CD ( 0.05)

b. Flowering time
Table 37 shows the day of first flowering of the plants Vigna radiata and
Vigna mungo grown in red soil and black soil using 25 % and 50% tannery
effluent.
TABLE 37
FLOWERING TIME OF PLANTS Vigna radiata AND Vigna mungo GROWN
IN RED SOIL AND BLACK SOIL USING DILUTED EFFLUENT
Soil
Groups
CR
Red soil
Black soil
Flowering time
Vigna radiata
Vigna mungo
45 4.08
45 4.04
T1R
47 0.82
47 1.63
T2R
49 0.80
49 0.86
CB
44 3.27
45 4.05
T1B
47 0.85
46 0.82
T2B
49 0.81
48 1.62
CD ( 0.05)
4.9

139
There was no significant delay in flowering time of Vigna radiata and

Vigna mungo plants grown using both the effluents in both the soils.
c. Pod length, Pod weight, Number of Pods/plant
Table 38 shows the pod length and pod weight of the plant of Vigna
tannery effluent.
Vigna radiata recorded significant (p<0.05) reduction in pod length grown
with black soil and 50% effluent. The pod weight of Vigna mungo was
decreased significantly with 50% effluent.
TABLE 38
POD LENGTH, POD WEIGHT OF Vigna radiata AND Vigna mungo
GROWN IN RED AND BLACK SOILS USING DILUTED
TANNERY EFFLUENT
Soil
Red
soil
Black
soil
CR
Pod length (cm)

Vigna
Vigna
radiata
mungo
4.70 0.16 1.42 0.02
Pod weight(g)
Vigna
Vigna
radiata
mungo
1.07 0.01
19.0 0.82
T1R
4.50 0.41
1.30 0.04
1.02 0.01
18.0 1.62
T2R
4.40 0.31
1.23 0.03
0.99 0.02
16.0 0.81
CB
4.50 0.43
1.31 0.01
1.02 0.03
18.0 0.80
T1B
4.10 0.09
1.28 0.02
0.99 0.02
17.0 0.84
T2B
3.90 0.05
0.99 0.01
0.81 0.03
15.0 0.86
Groups
CD ( 0.05)
0.5
2.6

Table 39 shows number of pods/ plant of Vigna radiata and Vigna mungo
grown in red soil and black soil using 25% and 50% tannery effluent.
A significant decrease was not observed in number of pods/plant in both the
plants and soils.
140
TABLE 39
NUMBER OF PODS / PLANT OF Vigna radiata AND Vigna mungo
GROWN IN RED AND BLACK SOILS USING DILUTED
TANNERY EFFLUENT
Soil
Groups
Red soil
Black soil
Pod length (cm)

Vigna radiata
Vigna mungo
CR
4.70 0.16
1.42 0.02
T1R
4.50 0.41
1.30 0.04
T2R
4.40 0.31
1.23 0.03
CB
4.50 0.43
1.31 0.01
T1B
4.10 0.09
1.28 0.02
T2B
3.90 0.05
0.99 0.01
CD ( 0.05)
0.5

d. Number of seeds / plant and Number of seeds /pod

Table 40 shows the number of seeds /plant and number of seeds /pod of
Vigna radiata and Vigna mungo plants grown in red soil and black soil using
25% and 50% tannery effluent.
The decrease in the number of seeds/ plant and number of seeds / pod in
effluent treated Vigna radiata and Vigna mungo plants grown in red and
black soils was not found to be significant compared to the control plants.
Sinha et al. (2008) demonstrated that V.radiata exhibited a significant increase
in growth parameters when grown on lower amendments of sludge.
141
TABLE 40
NUMBER OF SEEDS /PLANT AND NUMBER OF SEEDS /POD OF Vigna
radiata AND Vigna mungo GROWN IN RED AND BLACK SOILS
USING DILUTED TANNERY EFFLUENT
Soil
CR
Number of seeds /plant

Vigna
Vigna
radiata
mungo
219 0.82
113 2.43
Number of seeds /pod

Vigna
Vigna
radiata
mungo
13.0 2.45
8.0 0.81
T1R
213 2.46
109 0.83
12.0 1.63
7.0 0.80
T2R
202 1.62
106 0.85
11.0 0.87
6.0 0.88
CB
201 0.86
82 1.53
11.0 1.65
7.0 1.66
T1B
193 4.45
79 0.52
10.0 0.72
5.0 0.89
T2B
185 4.08
72 1.61
10.0 1.67
6.0 0.81
Groups
Red
soil
Black
soil
CD ( 0.05)
2.02
2.75

CB- Control black soil, T1B- Black soil with 25% effluent, T2B- Black soil with 50% effluent.
e. Total seed weight / plant and 100 seed weight

Table 41 shows the total seeds weight / plant and 100 seeds weight of
Vigna radiata and Vigna mungo plants grown in red soil and black soil in 25%
Total seeds weight / plant of Vigna radiata and Vigna mungo showed no
significant reduction when grown in 25% and 50% effluent. This indicates that
the diluted tannery effluent might be used for plant growth. 100 seed weight of
both the plants were found to be significantly decreased with 50% effluent in
both the soils.
Results of yield parameters of both the plants grown in red soil and black
soil in both the effluent concentrations indicated that plants grow better in 25%
tannery effluent and diluted effluent could be a better choice for plant growth in
industrialized area.
142
TABLE 41
TOTAL SEED WEIGHT/ PLANT AND 100 SEED WEIGHT OF Vigna radiata
AND Vigna mungo GROWN IN RED AND BLACK SOILS USING
Soil
Groups
Red
soil
Black
soil
Total seed weight/ plant (g)

Vigna
Vigna
radiata
mungo
100 seed weight (g)

Vigna
Vigna
radiata
mungo
CR
16.40 0.33
45.0 4.08
7.60 0.33
4.51 0.01
T1R
15.70 0.06
47.0 0.82
7.40 0.28
4.21 0.03
T2R
14.30 0.24
49.0 0.80
7.10 0.16
3.80 0.17
CB
15.80 0.18
44.0 3.27
7.90 0.08
4.60 0.15
T1B
14.20 0.16
47.20 0.85
7.40 0.35
4.20 0.18
T2B
13.70 0.13
49.0 0.81
6.90 0.07
4.0 0.41
CD ( 0.05)
2.02
2.75

Hence, from the results of this phase it was found that the biometric
observations and biochemical parameters such as chlorophyll, carbohydrate,
carotenoid, ascorbic acid and yield parameters of the plants grown in red soil
were at higher levels compared to those grown in black soil. Vigna radiata
showed a better response with respect to biometric observations and certain
biochemical parameters compared to Vigna mungo. Plants grown with 25%
effluent exhibited a better growth compared to those grown with 50% effluent.
PHASE III
4.3. Health profile of Tannery workers
Any effort to evaluate occupational health risks includes assessing the
health of individual workers with the goal of keeping the worker healthy and
reducing the overall risks in the work environment.
143
A worker is at risk if he/she has a greater chance of developing disease

than a non exposed worker. It is very important to identify all harmful
substances and to monitor and control exposures in order to manage the risk
(Hall, 2001).
Leather production includes many operations with different exposures,
which can be harmful for the health of the workers and particularly be
carcinogenic (Issever et al., 2007). Certain chemicals such as benzene based
dyes and formaldehyde are considered to be carcinogenic (Budhwar, 2005).
Besides these, scores of other chemicals and organic solvents such as
chromate and bichromate salts, aniline, butyl acetate, ethanol, benzene,
toluene, suplhuric acid and ammonium hydrogen sulfide are used in the
tannery industry. An important health risk factor for the tannery workers is
occupational exposure to chromium mainly in organic form or in protein bound
form caused by leather dust (Mikoczy and Hagmar, 2005). Chromium may
enter the body by inhalation, ingestion and by direct cutaneous contact.
Professional exposure to chromium increases the risk of dermatitis, ulcers and
perforation of the nasal septum and respiratory illness as well as increased lung
and nasal cancers. Chromium specific health hazards like carcinoma of the
larynx and lung parenchyma and paranasal sinusis have also been reported
(Rastogi et al., 2007).
According to Bulletin of the WHO (2005), 58% of the tannery workers
were found to suffer from gastrointestinal diseases, 31% from dermatological
diseases, 12% from hypertension and 0.9 % from jaundice.
Working conditions, nature of work, vocational and professional status
and geographical location of industries and employment have a profound
impact on the social status and social well being of the workers (Babalola and
Babajide, 2009)
Exposure is the contact of toxic substance by the body. It may be acute
(immediate) or chronic (long term). During leather tanning, the workers are
144
exposed to chemicals such as sodium chloride during soaking stage, sulphide

and lime in fleshing and trimming stage, acid and ammonium salts in bating
stage, fungicides and bactericides in picking stage, chromium salts during
tanning stage, nickel, arsenic, zinc, cadmium, copper, dye, and solvents
during wet finishing stage. The chemicals used in the process of tanning have
been proved to be toxic.People involved in producing leather have a
significant risk of presenting clinical conditions attributed to chromium exposure
(Cuberos et al., 2009).
The use of sulphides and hydrosulphides in dehairing operations may

carry a risk of skin contact for the operator. A similar risk is possible in the use of
caustic materials such as sodium hydroxide or calcium hydroxide causing skin
burns. Formaldehyde, glutaraldehyde and hydrogen peroxide used in tanning
processes causes irritation of body tissues even in if minute quantities are
inhaled. A variety of dyestuffs and fungicides are used which are quite serious if
inhaled or injested (Taylor et al., 2006).
Continuous exposure to these chemicals results in entry into the body.
Workers who have been dealing with these chemicals and who do not follow
any safety measures in preventing the entry of the chemicals into the body,
were found to acquire many forms of ill effects. Eating in contaminated area,
using the chemicals with bare hand, breathing without using mask in the
workplace are some of the reasons of the entry of these hazardous chemicals.
Hence in this phase of the present study an attempt was made to analyze
the biochemical parameters associated with hepatic, renal and skin disorders
caused, if any in the selected group tannery workers who have been exposed to
these chemicals for many years and who do not follow any safety measures
while using the chemicals.
Tannery workers with one to five years of experience (Group II: n=20)
and workers with five to ten years of experience (Group III : n= 20), were
145
selected for the study. A reference group of 20 subjects belonging to the similar
age group who never had any occupational exposure in the tanneries served as
control (Groiup I). Each group comprised of 60% men and 40% women workers
who belong to the same socio economic group and dietary pattern. They
belonged to an age group of 20-50 years and they were under cumulative
exposure to numerous pollutants in the workplace. They had no history of
defect, infections or metabolic disorders. These workers were not engaged in
any other occupation and hence not exposed to other types of pollutants.
Hematological
parameters
(hemoglobin,
total
count
and
immunoglobulin E), assessment of liver function (alanine transaminase,

aspartate transamiase, alkaline phosphatase, acid phosphatase and lactate
dehydrogenase), assessment of renal function (urea, uric acid and creatinine)
and mineral status (chromium, nickel, zinc and cadmium) of the selected
tannery workers were performed and their results are discussed as follows.
4.3.1 Hematological parameters of blood sample
Hematological parameters are related to the changing environmental
conditions and therefore can be used to screen the health state of organisms
exposed to a particular toxicant (Tripathi et al., 2002).
4.3.1.1. Hemoglobin and total leukocyte count
The mean values of hemoglobin and total leukocyte count estimated in
control and experimental groups are presented Table 42 and Figure 28 and
Figure 29.
The normal hemoglobin level is 12-14 g/dl. Group I participants
(control) had hemoglobin contents within the normal limits, whereas group II and
group III recorded significantly lower levels than the control. The total count of
the experimental groups was found to be increased than that of the control
groups.
146
TABLE 42
HEMOGLOBIN CONTENT AND TOTAL LEUKOCYTE COUNT
OF THE TANNERY WORKERS
Groups
Hemoglobin
(g/100 ml)
Total leukocyte
count (mm3)
Group I
Control
12.33 0.08
10,100 105.7
Group II
1-5 years
9.60 0.7
11,000 104.9
Group III
5-10 years
7.87 0.9
12,320 96.2
CD (0.05)
0.19
112.57
Values are presented as mean SD (n = 20 in each group)
The
reduction
FIG. 28 HEMOGLOBIN CONTENT
16
be due to the effect of
14
pollutants on hematopoietic
system
anemic
which
leads
condition
to
in
human (Mathivanan, 2004).

Hematological values such
Hemoglobulin (g/100ml)
of hemoglobin content might
12
10
8
6
4
2
0
Group I
Group II
Group III
as white blood cell count and

red blood cell count of population residing in industrial area exposed to toxic
Total leucocyte count (mm 3 )
14000
FIG. 29 TOTAL LEUKOCYTE COUNT
12000
pollutants
showed
increased
trend
the
10000
hemoglobin
while
content
8000
decreased
6000
population
4000
the
2000
population (Ahsan, 2003).
non
in
an
exposed
compared
to
exposed
According to Benova et al.
0
Group I
Group II
Group III
(2002), chromium is rapidly
147
absorbed by the lungs into the blood and easily penetrates the cellular
membranes and binds to the hemoglobin in the red blood cells, thereby affecting
the oxygen carrying capacity and impairing the lung function status.
Elevated leucocyte count might be due to increase in the population
of neutrophils, acidophils and basophils (Joshi et al., 2002). Cases of
hematological effects have been reported in humans after the ingestion of lethal
or sublethel doses of chromium. Decreased hemoglobin (anemia) content and
increased total white blood cell count (thrombocytopenia) were noted by
Parveen and Rawat (2010). Alterations in blood hemoglobin, total cell count
and erythrocyte sedimentation have provided a useful means of detecting
and assessing the severity of anemia due to exposure to toxic pollutants
(Sharma et al., 2004).
4.3.1.2 Immunoglobulin E levels
Table 43 and Figure 30 shows the Immunoglobulin E levels in blood
sample of the participants.
TABLE 43
Ig E LEVELS OF THE WORKERS
Groups
IgE (IU/L)
Group I
98.6 1.3
Group II
732 1.9
Group III
800 2.3
CD ( 0.05)
4.8
The results show that both the experimental groups had significantly
increased levels of Immunoglobin E compared to the Group I (control)
participants. For any allergic reactions, elevated Ig E is an important
determinant. Occupational exposure and elevated serum Ig E levels were well
correlated in the study conducted by Kim et al., (2010).
148
Sensitizers are agents

Immunoglobulin (IU / L)
900
FIG. 30 Ig E LEVELS OF THE WORKERS
800
that may cause allergic or
700
allergic-like
responses
to
600
occur.
500
After
an
initial
exposure to a substance an
400
300
individual
200
may
become
sensitized to that substance.
100
0
Group I
Group II
Group III
Subsequent
exposures
to
the same substance, often at

a much lower concentration than before, produce an allergic response. This
response may be a skin rash (dermatitis) or an asthmatic-like attack, depending
on the route of exposure (Shahzad et al., 2008).
Leather tanning is principally chemical preservation of raw hide by the
process in which binding of various chemicals (mainly chromium salts as
potassium dichromate) to proteins in raw hide takes place. Chromium has
potential to bind with skin proteins of tannery workers to produce complex
antigens which lead to hypersensitivity. The resulting contact dermatitis could be
preliminary condition to the development of bronchial asthma (Lockman, 2002).
Tannery workers are thus potentially exposed to harmful agents,
rendering them vulnerable to health problems especially those of skin and
gastrointestinal problems. Due to this exposure, health hazards namely eye
irritation and repiratory tract irritation would be caused. Skin ulcer might develop
if chromium compounds come into contact with an abrasion, a scratch of
laceration of the skin (Shahzad et al., 2006).
In the present study, 12 out of 20 persons of groups III who had more
years of exposure were found to possess skin rashes and scales indicating
allergic response. All the respondents were associated with chrome tanning
process. They handle lead chromate and nickel chromate every day. And their
contact with these chemicals occurs for more than 6 hours a day. The route of
149
entry was mainly absorption by skin contact since they seldom wore gloves
while operating the chemicals. Other possible routes of entry were inhalation of
chrome dust and ingestion since they have lunch in the work place itself without
proper handwash. Repeated contact with toxic metals might be the cause for
allergic contact dermatitis.
Plate 9 shows a person affected by dermatitis. He was associated with all
the processes of tanning since he has 18 years of experience in tannery
industry. His Ig E levels were found to be increased than the normal limits and
had skin allergy. This person was associated with all the process of tanning. He
handled chemicals namely chrome, formate and potassium dichromate daily.
PLATE 9
DERMATITIS LIKE SYMPTOM
In a study conducted by Rastogi et al. (2008), the leather tanners who

had a mean exposure of 8 years in the tanneries were found to have
dermatological diseases such as rashes and papules along with complaints of
itching. The burning sensation was also reported by 15 subjects in the exposed
workers. Chromium toxicity can produce penetrating lesions known as chrome
hole or chrome ulcers particularly in areas where a break in the epidermis is
150
already present. These commonly occur on the fingers, knuckles, and fore
arms. The characteristic chrome sore begins as a papule forming an ulcer with
raised hard edges. Ulcers can penetrate deep into soft tissue or become the
sites of secondary infection (Meditext, 2005). Chrome ulceration is a specific
skin lesion caused as a result of direct contact with trivalent or hexavalent
chromium compounds and is especially observed among chrome tanners.
The affected workman has painless, multiple ulcers of holes on the skin
of the exposed parts of the body, especially hands and feet. In a study
conducted in North India, the prevalence of ulcers of fingers and toes among
chrome tanners was found to be 10.6% (Raidas, 2007).
The most viable condition that aggravates the risk of developing
dermatitis is the constant wetting of the skin. Persons who are engaged in
soaking operations were found to be maximum affected (10.46%) with
dermatitis.
4.3.2. Assessment of liver function
Clinical laboratories use the measurement of alanine transaminase
(ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) , acid
phosphatase (ACP) and lactate dehydrogenase (LDH) in serum and tissue for
assessing liver damage (Karthikeyan et al., 2004).
Table 44, Figures 31 and 32 shows the activities of the liver enzymes
alanine transaminase, aspartate transaminase, alkaline phosphatase, acid
phosphatase and lactate dehydrogenase of tannery workers
The normal value of ALT and AST were up to 40 IU/L and 38 IU/L
respectively. The control group recorded a value within the normal range,
whereas ALT of group III workers showed a significant increase and AST
activity of group II and group III workers showed a significant increase.
151
TABLE 44
ALANINE TRANSAMINASE (ALT), ASPARTATE TRANSAMINASE (AST),
ALKALINE PHOSPHATASE (ALP), ACID PHOSPHATASE (ACP)
AND LACTATE DEHYDROGENASE (LDH) ACTIVITIES
OF THE TANNERY WORKERS
GROUPS
ALT (IU/L)
AST (IU/L)
ALP (IU/L)
ACP (IU/L)
LDH (IU/L)
Group I
38.200.78
34.15 0.01
32.90.9
4.780.01
1620.54
Group II
41.200.17
38.580.98
56.80.5
3.630.06
2410.62
Group III
42.360.42
39.430.84
83.20.4
3.870.08
2860.71
CD (0.05)
1.10
0.16
0.16
0.11
1.99

ALT
AST
ALP
ACP
LDH
- Activity of enzymes that transforms 1 mol of the L.alanine in 1 min to pyruvate

- Activity of enzymes that transforms 1 mol of the aspartate in 1 min to pyruvate
- Activity of enzyme that converts 1 mg phenol in 1 minute in 100 ml of serum
- Moles of p-nitrophenol released/ min/mg protein
- The activity which produces a change in extinction of 0.001/ min
FIGURE 31
ALANINE TRANSAMINASE AND ASPARTATE TRANSAMINASE
ACTIVITIES OF THE TANNERY WORKERS
45
40
35
IU / L
30
25
20
15
10
5
0
Alanine transaminase
Aspartate transaminase
152
FIGURE 32
ALKALINE PHOSPHATASE, ACID PHOSPHATASE AND LACTATE
DEHYDROGENASE ACTIVITIES OF THE
TANNERY WORKERS
90
ALKALINE PHOSPHATASE
80
ALP (IU / L)
70
60
50
40
30
20
10
0
Group I
Group II
Group III
ACID PHOSPHATASE
ACP (IU / L)
5
4
3
2
1
0
Group I
350
Group II
Group III
LACTATE DEHYDROGENASE
LDH (IU / L)
300
250
200
150
100
50
0
Group I
Group II
Group III
153
Group II and group III participants showed a significant increase in

alkaline phosphatase and lactate dehydrogenase activities whereas acid
phosphatase activity of group II and group III participants decreased significantly
compared to the group I participants.
Increase in serum aminotransferase levels characterizes cirrhosis,
cholestatic liver disease, fatty liver and hepatic neoplasms (Das and
Vasudevan, 2005). Gupta et al. (2005) and Johri et al.(2004) reported that rise
in aspartate aminotransferase and alanine aminotransferase is indicative of
leakage of enzymes from liver resulting in alterations in the cell permeability.
Chromium has been reported to cause severe hepatic effects, elevated liver
enzyme levels, hepatomegaly and hepatic failure in four of five workers exposed
to chromium in leather tanning industry (Munagala et al., 2003). The present
study falls in line with the work of Yun et al. (2008) who reported a significant
increase in serum LDH level in tannery industry workers when compared to
control subjects.
LDH serves as a sensitive marker of epidermal toxicity of stress induced
by pathological conditions and environmental conditions (Sharma et al., 2010)
Its level is increased in persons with anemia, leukemia, urinary tract infection
and pulmonary embolism reflecting its diagnostic utility in specific clinical cases
(Nussinovitch et al., 2009; Shahi et al., 2009).
4.3.3. Assessment of renal function
In order to see the long term effects of pollutants on kidney different renal
indices were measured in both exposed and non exposed groups and the
results are presented.
Tables 45, 46 and Figure 33 indicate the urea, uric acid and creatinine
contents of the serum sample and urine sample of the participants respectively.
The normal blood urea level is 15-45 mg/dl, uric acid is up to 4.8 mg/dl and
creatinine is 0.7 to 1.4 mg/dl. In our study, all the three groups recorded normal
values of urea, uric acid and creatinine contents in the blood sample.
154
TABLE 45
UREA, URIC ACID AND CREATININE LEVELS IN THE BLOOD
SAMPLE OF THE TANNERY WORKERS
Groups
Urea
(mg/dl)
Uric acid
(mg/dl)
Creatinine
(mg/dl)
Group I
26.39 0.26
3.29 0.09
0.94 0.02
Group II
30.35 0.29
3.97 0.08
1.10 0.06
Group III
37.70 0.31
4.26 0.05
1.29 0.07
CD (0.05)
0.19
0.18
0.02
TABLE 46
UREA, URIC ACID AND CREATININE LEVELS IN THE URINE
SAMPLE OF THE TANNERY WORKERS
Groups
Urea
(g/day)
Uric acid
(g/day)
Creatinine
(g/day)
Group I
12.201.4
0.410.05
2.520.11
Group II
14.201.1
0.480.02
2.710.28
Group III
15.101.02
0.530.06
3.010.19
CD (0.05)
0.18
0.02
0.21
155
FIGURE 33
UREA, URIC ACID AND CREATININE CONTENTS IN THE BLOOD AND
URINE SAMPLES OF THE TANNERY WORKERS
BLOOD SAMPLE
40
URINE SAMPLE
18
UREA
14
Urea (g/day)
Urea (mg/dl)
30
25
20
15
10
12
10
8
6
4
0
Group I
Group II
Group III
Group I
URIC ACID
Group II
Group III
URIC ACID
0.7
4.5
0.6
Uric acid (g/day)
4
Uric acid (mg/dl)
UREA
16
35
3.5
3
2.5
2
1.5
0.5
0.4
0.3
0.2
1
0.1
0.5
0
Group I
Group III
Group I
3.5
CREATININE
1.6
1.4
1.2
2.5
Creatinine (g/day)
Creatinine (mg/dl)
Group II
1
0.8
0.6
0.4
Group II
Group III
CREATININE
2
1.5
1
0.5
0.2
0
Group I
Group II
Group III
Group I
Group II
Group II
156
Similarly, urea, uric acid and creatinine contents in urine were well within the
normal limits indicating that exposure to pollutants had no ill effect on the renal
function. This was supported by the findings of Orisakwe et al.(2007), who found
no detectable differences in the renal indices like urea, uric acid and creatinine
contents between exposed and non exposed groups.
3.5. Metal contents in the tannery workers
Heavy metals namely zinc, cadmium and lead are considered to be the
frequent pollutants of natural environments causing serious health hazards
(Mathad et al., 2004). They are generally deposited in liver, muscle, kidney,
spleen, skin, bone and soft tissues of human being. USEPA (United States
Environmental Protection Agency) considers eight trace metals as high priority
critical metals namely arsenic, cadmium, copper, chromium, lead, mercury,
nickel and zinc (Sarker and Gupta, 2003).
Environment pollution by compounds of heavy metals is increasing with
extensive industrial developments. Large amounts of chromium were introduced
into the environment through tannery, textile, chemical manufacture, metal
plating and many other industrial effluents thus making them available to
plants, animals and humans (Raj and Raghavan, 2002). More than 170,000
tons of chromium wastes are discharged to the environment annually as a
consequence of industrial and manufacturing activities (Kamaludeen et al.,
2003). Zinc as a trace element, is regarded as an essential nutrient for human
beings and has also been found to be protective in some kind of liver injury.
Alcoholic cirrhosis may be associated with a state of zinc deficiency (Dhawan
et al., 2005). It is an important component of several enzymes and nutrients.
The water having more than 5mg/L of zinc gives an undesirable astringent taste
and is unsuitable for drinking and cooking purposes (Rani and Reddy, 2003).
The various production processes in tanning industry pose many hazards
to the health of its employees. Toxic chemicals such as hydrogen sulphide,
chromium, bleaching agent, disinfectants, dyes and physical and biological
157
agents are a few to mention. Their effects vary from minor irritation while
working to serious and disabling diseases (Raidas, 2007).
In the present study, to find out the toxicological aspects of the exposed
metals to human beings blood and urine samples were analyzed for the metal
contents namely chromium, nickel, zinc and cadmium.
Table 47 and Figure 34 indicate the chromium, nickel, zinc and cadmium
contents of blood samples and Table 48 indicates that of urine samples of the
participants respectively.
TABLE 47
CHROMIUM, NICKEL, ZINC AND CADMIUM LEVELS IN THE
BLOOD SAMPLE OF THE PARTICIPANTS
Groups
Chromium
Nickel
Zinc
Cadmium
mg/l
Group I
0.0100.001
0.0070.00
0.0160.001
0.0160.003
Group II
0.0190.002 0.0190.001
0.0160.002
0.0190.002
Group III
0.0320.002 0.0190.001
0.0260.002
0.0260.001
0.006
0.003
CD ( 0.05)
0.002
0.001
TABLE 48
CHROMIUM, NICKEL ZINC AND CADMIUM LEVELS IN THE
URINE SAMPLE OF THE PARTICIPANTS
Groups
Chromium
Nickel
Zinc
Cadmium
mg/l
Group I
0.010.001
ND
ND
ND
Group II
0.010.001
ND
ND
ND
Group III
0.020.001
ND
ND
ND
CD ( 0.05)
0.002

ND- Not detectable
158
FIGURE 34
CHROMIUM, NICKEL, ZINC AND CADMIUM CONTENTS
OF THE BLOOD SAMPLE
0.04
Group I
Group II
Group III
0.035
0.03
mg / l
0.025
0.02
0.015
0.01
0.005
0
Chromium
Nickel
Zinc
Cadmium
Chromium levels in the blood of the control group were found to be

0.01 mg/l which was significantly increased (0.019 and 0.032 mg/l) in group II
and group III participants. Chromium level in urine was found to be 0.01 mg/l.
The value was significantly increased in group III participants. Chromium is toxic
and mutagenic to most organisms and is known to cause irritation, corrosion of
the skin and respiratory tract; it also causes lung carcinoma in humans (Ganguli
and Tripathi 2002). Rastogi et al. (2008) reported that the urinary and blood
concentrations of chromium were found to be significantly raised among the
leather tanners thereby reflecting the bodys burden of chromium in the exposed
workers as a result of a high concentration of environmental chromium at the
work place.
An important health risk factor for the tannery workers is occupational
exposure to chromium which is used as a basic tanning pigment. The workers
on exposure to leather dust which contains chromium in the protein bound form
exhibited a higher mean concentration of urinary and blood chromium. The
lungs, intestinal tract, the liver and the kidney are the target organs for
159
chromate toxicity (Rom, 2007). Higher values of blood and urinary chromium
concentration were observed in the tannery workers when compared with
healthy normal adult values as reported by Kornhauser et al. (2002).
Nickel content in the blood sample was found to be 0.007 mg/l in control
group which increased to 0.019 in both the group. There was no detectable
nickel in the urine sample. Nickel is the potential carcinogen for lung and may
cause allergies, lung fibrosis and cancer of respiratory tract in occupationally
exposed populations (Kazprazak et al., 2003). Nickel used as nickel carbonate
in wet finishing and pickling processes of leather making might be the cause of
increased nickel concentration in the blood sample of tannery workers.
The zinc content of the control and group II sample remained the same
ie. 0.016 mg/l which was increased to 0.026 mg/l in group III. Zinc was not
detectable in the urine sample. Acute zinc toxicity in humans includes vomiting,
dehydration, drowsiness, lethargy, nausea, lack of muscular co-ordination, and
renal failure. Workers exposed to zinc fumes from smelting or welding have
suffered from a short term illness called mental fern-fever (Leghouchi et al.,
2008).
Zinc hydrosulfate and zinc sulfite were used in the process of tanning
instead of wet blue which was banned after 1990. Workers exposed to hides
treated with zinc chloride, zinc hydroxide and zinc sulfite were found to have a
higher zinc content in blood sample due to their frequent handling.
Cadmium content of the blood sample of group I participants was
0.016 mg/l whereas group II and group III showed a value of 0.019 and
0.026 mg/l. the content was not detectable in urine sample. Prolonged exposure
to cadmium can cause yellow stain that gradually appears on necks and teeth
(Meena et al., 2004). Cadmium induced adverse health effects in humans were
reported by Pizent et al. (2003).
Heavy metals affect individuals at sublethal concentration by changing
the activities of key biomolecules. A chronic dose of zinc, lead, copper,
160
cadmium, nickel and chromium increases the risk of developing anemia,

damage to pancreas, lowers down HDL cholesterol levels and raises
LDL cholesterol levels and possibly enhances the symptoms of the Alzhemiers
disease, hepatic and renal damage (Sharma and Agrawal, 2005). The
excessive intake of metal by man leads to severe mucosal irritation, widespread
capillary damage, hepatic and renal damage, central nervous problems followed
by depression, gastrointestinal irritation and possible necrotic changes in the
liver and kidney (Kalavathy et al., 2005).
Hence, the clinical study indicates that the workers with greater years of
exposure had a risk of dermatitis like symptom due to unsafe handling of
chemicals.
161

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Results and Discussion

RESULTS AND DISCUSSION

Results and Discussion

Results and Discussion

parameters. The results of the physical parameters of the raw effluent

Results and Discussion

Total hardness (mg/l)

Electrical conductivity (mg/l)

Results and Discussion

pH was recorded as 10.5, which was above the tolerance limit of

reported that larger solid particulate matter remains suspended as a result of

Results and Discussion

Chemical Oxygen Demand

Biological Oxygen Demand

Values are mean of triplicates

Results and Discussion

Chemical oxygen demand (COD) is the test used to measure pollution

Results and Discussion

Results and Discussion

present in effluent. In a similar study done by Zahid et al. (2006), extremely

Results and Discussion

4.1.2. Biochemical profile of the target area water samples

Results and Discussion

Target area water

Electrical conductivity (mho/cm)

Total suspended solids

Total dissolved solids

Values are mean of triplicates

pH and Electrical conductivity (EC)

Results and Discussion

The higher values of EC could be the result of extreme concentrations of soluble

Results and Discussion

Total hardness, calcium and magnesium hardness

Results and Discussion

Organization (2005), the metals of most immediate concern are aluminum,

Results and Discussion

application of raw sewage effluent results in the accumulation of these nutrients

Results and Discussion

Values are mean of triplicates

Soil pH is one of the most influential parameters controlling the

Results and Discussion

Results and Discussion

CR - Control red soil

TR - Target area red soil

CB - Control black soil

TB - Target area black soil

Results and Discussion

CR - Control red soil

TR - Target area red soil

CB - Control black soil

TB - Target area black soil

Results and Discussion

CR - Control red soil

TR - Target area red soil

CB - Control black soil

TB - Target area black soil

Results and Discussion

was higher than the control black soil.

Increased amount of iron may be

Results and Discussion

Presence of chromium and nickel in soil disturbs the pattern of

nutrient uptake by increasing or decreasing the nutrient content in plants

CR - Control red soil

TR - Target area red soil